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Sample records for factor xa inhibitor

  1. Anticoagulation by factor Xa inhibitors

    PubMed Central

    Orfeo, Thomas; Butenas, Saulius; Brummel-Ziedins, Kathleen E.; Gissel, Matthew; Mann, Kenneth G.

    2010-01-01

    Summary Background Therapeutic agents that regulate blood coagulation are critical to the management of thrombotic disorders, with the selective targeting of factor (f)Xa emerging as a promising approach. Objective To assess anticoagulant strategies targeting fXa. Methods A deterministic computational model of tissue factor (Tf)-initiated thrombin generation and two empirical experimental systems (a synthetic coagulation proteome reconstruction using purified proteins and a whole blood model) were used to evaluate clinically relevant examples of the two available types of fXa directed anticoagulants (an antithrombin (AT)-dependent agent, fondaparinux, and an AT-independent inhibitor, Rivaroxaban) in experimental regimens relevant to long term (suppression of new Tf-initiated events) and acute (suppression of ongoing coagulation processes) clinical applications. Results Computational representations of each anticoagulant’s efficacy in suppressing thrombin generation over a range of anticoagulant concentrations in both anticoagulation regimens were validated by results from corresponding empirical reconstructions and were consistent with those recommended for long term and acute clinical applications respectively. All three model systems suggested that Rivaroxaban would prove more effective in the suppression of an ongoing coagulation process than fondaparinux, reflecting its much higher reactivity toward the prothrombinase complex. Conclusion The success of fondaparinux in acute settings in vivo is not explained solely by its properties as an fXa inhibitor. We have reported that fIXa contributes to the long term capacity of clot-associated catalysts to restart a coagulation process [Orfeo T et al. J Biol Chem 2008;283:9776], suggesting that the enhanced anti-fIXa activity of fondaparinux-AT may be critical to its success in acute settings in vivo. PMID:20492473

  2. Phenyltriazolinones as potent factor Xa inhibitors.

    PubMed

    Quan, Mimi L; Pinto, Donald J P; Rossi, Karen A; Sheriff, Steven; Alexander, Richard S; Amparo, Eugene; Kish, Kevin; Knabb, Robert M; Luettgen, Joseph M; Morin, Paul; Smallwood, Angela; Woerner, Francis J; Wexler, Ruth R

    2010-02-15

    We have discovered that phenyltriazolinone is a novel and potent P1 moiety for coagulation factor Xa. X-ray structures of the inhibitors with a phenyltriazolinone in the P1 position revealed that the side chain of Asp189 has reoriented resulting in a novel S1 binding pocket which is larger in size to accommodate the phenyltriazolinone P1 substrate.

  3. Engineered factor Xa variants retain procoagulant activity independent of direct factor Xa inhibitors.

    PubMed

    Verhoef, Daniël; Visscher, Koen M; Vosmeer, C Ruben; Cheung, Ka Lei; Reitsma, Pieter H; Geerke, Daan P; Bos, Mettine H A

    2017-09-13

    The absence of an adequate reversal strategy to prevent and stop potential life-threatening bleeding complications is a major drawback to the clinical use of the direct oral inhibitors of blood coagulation factor Xa. Here we show that specific modifications of the substrate-binding aromatic S4 subpocket within the factor Xa active site disrupt high-affinity engagement of the direct factor Xa inhibitors. These modifications either entail amino-acid substitution of S4 subsite residues Tyr99 and/or Phe174 (chymotrypsinogen numbering), or extension of the 99-loop that borders the S4 subsite. The latter modifications led to the engineering of a factor Xa variant that is able to support coagulation in human plasma spiked with (supra-)physiological concentrations of direct factor Xa inhibitors. As such, this factor Xa variant has the potential to be employed to bypass the direct factor Xa inhibitor-mediated anticoagulation in patients that require restoration of blood coagulation.A major drawback in the clinical use of the oral anticoagulants that directly inhibit factor Xa in order to prevent blood clot formation is the potential for life threatening bleeding events. Here the authors describe factor Xa variants that are refractory to inhibition by these anticoagulants and could serve as rescue agents in treated patients.

  4. Factor Xa: Simulation studies with an eye to inhibitor design

    NASA Astrophysics Data System (ADS)

    Daura, Xavier; Haaksma, Eric; van Gunsteren, Wilfred F.

    2000-08-01

    Factor Xa is a serine protease which activates thrombin and plays a key regulatory role in the blood-coagulation cascade. Factor Xa is at the crossroads of the extrinsic and intrinsic pathways of coagulation and, hence, has become an important target for the design of anti-thrombotics (inhibitors). It is not known to be involved in other processes than hemostasis and its binding site is different to that of other serine proteases, thus facilitating selective inhibition. The design of high-affinity selective inhibitors of factor Xa requires knowledge of the structural and dynamical characteristics of its active site. The three-dimensional structure of factor Xa was resolved by X-ray crystallography and refined at 2.2 Å resolution by Padmanabhan and collaborators. In this article we present results from molecular dynamics simulations of the catalytic domain of factor Xa in aqueous solution. The simulations were performed to characterise the mobility and flexibility of the residues delimiting the unoccupied binding site of the enzyme, and to determine hydrogen bonding propensities (with protein and with solvent atoms) of those residues in the active site that could interact with a substrate or a potential inhibitor. The simulation data is aimed at facilitating the design of high-affinity selective inhibitors of factor Xa.

  5. A specific antidote for reversal of anticoagulation by direct and indirect inhibitors of coagulation factor Xa.

    PubMed

    Lu, Genmin; DeGuzman, Francis R; Hollenbach, Stanley J; Karbarz, Mark J; Abe, Keith; Lee, Gail; Luan, Peng; Hutchaleelaha, Athiwat; Inagaki, Mayuko; Conley, Pamela B; Phillips, David R; Sinha, Uma

    2013-04-01

    Inhibitors of coagulation factor Xa (fXa) have emerged as a new class of antithrombotics but lack effective antidotes for patients experiencing serious bleeding. We designed and expressed a modified form of fXa as an antidote for fXa inhibitors. This recombinant protein (r-Antidote, PRT064445) is catalytically inactive and lacks the membrane-binding γ-carboxyglutamic acid domain of native fXa but retains the ability of native fXa to bind direct fXa inhibitors as well as low molecular weight heparin-activated antithrombin III (ATIII). r-Antidote dose-dependently reversed the inhibition of fXa by direct fXa inhibitors and corrected the prolongation of ex vivo clotting times by such inhibitors. In rabbits treated with the direct fXa inhibitor rivaroxaban, r-Antidote restored hemostasis in a liver laceration model. The effect of r-Antidote was mediated by reducing plasma anti-fXa activity and the non-protein bound fraction of the fXa inhibitor in plasma. In rats, r-Antidote administration dose-dependently and completely corrected increases in blood loss resulting from ATIII-dependent anticoagulation by enoxaparin or fondaparinux. r-Antidote has the potential to be used as a universal antidote for a broad range of fXa inhibitors.

  6. Edoxaban: a new oral direct factor xa inhibitor.

    PubMed

    Camm, A John; Bounameaux, Henri

    2011-08-20

    Edoxaban is an oral direct factor Xa inhibitor that is currently undergoing investigation in phase III clinical trials for the prevention of stroke in patients with atrial fibrillation (AF) and for the prevention and treatment of venous thromboembolic events (VTE). Factor Xa is an attractive target for anticoagulant treatment, as it is the primary and rate-limiting source of amplification in the coagulation cascade. Edoxaban is a competitive inhibitor of factor Xa and has >10 000-fold greater selectivity for factor Xa relative to thrombin. In phase I clinical trials, the anticoagulant effects of edoxaban included dose-dependent increases in activated partial thromboplastin time and prothrombin time following single edoxaban doses of 10-150 mg and after multiple ascending doses (60 mg twice daily, 90 mg daily and 120 mg daily). The anticoagulant effects of edoxaban were rapid in onset (time to peak plasma concentration 1-2 hours) and sustained for up to 24 hours. Prolongation of bleeding time in 8% of subjects was >9.5 minutes (none of which appeared to be clinically significant) 2 hours after initial dosing, and was independent of edoxaban dose, formulation or dietary state. In general, plasma edoxaban concentrations were linearly correlated with coagulation parameters. Phase II clinical trials in patients with AF and VTE suggest that the edoxaban 30 mg once-daily and 60 mg once-daily regimens had a similar or better safety profile compared with dose-adjusted warfarin (international normalized ratio 2.0-3.0) in terms of bleeding events, and that edoxaban was not associated with hepatotoxicity. In addition, edoxaban was associated with statistically significant dose-dependent reductions in VTE after orthopaedic surgery compared with placebo or dalteparin sodium. Further clinical investigation of the efficacy and safety of once-daily edoxaban is being conducted in phase III clinical trials in comparison with warfarin in patients with AF in the phase III

  7. Design of novel aminopyrrolidine factor Xa inhibitors from a screening hit.

    PubMed

    Zbinden, Katrin Groebke; Anselm, Lilli; Banner, David W; Benz, Jörg; Blasco, Francesca; Décoret, Guillaume; Himber, Jacques; Kuhn, Bernd; Panday, Narendra; Ricklin, Fabienne; Risch, Philippe; Schlatter, Daniel; Stahl, Martin; Thomi, Stefan; Unger, Robert; Haap, Wolfgang

    2009-07-01

    Starting from a hit identified by focused screening, 3-aminopyrrolidine factor Xa inhibitors were designed. The binding mode as determined by X-ray structural analysis as well as the pharmacokinetic behaviour of selected compounds is discussed.

  8. Kinetic characterization of factor Xa binding using a quenched fluorescent substrate based on the reactive site of factor Xa inhibitor from Bauhinia ungulata seeds.

    PubMed

    Oliva, M L V; Andrade, S A; Juliano, M A; Sallai, R C; Torquato, R J; Sampaio, M U; Pott, V J; Sampaio, C A M

    2003-07-01

    The specific Kunitz Bauhinia ungulata factor Xa inhibitor (BuXI) and the Bauhinia variegata trypsin inhibitor (BvTI) blocked the activity of trypsin, chymotrypsin, plasmin, plasma kallikrein and factor XIIa, and factor Xa inhibition was achieved only by BuXI (K(i) 14 nM). BuXI and BvTI are highly homologous (70%). The major differences are the methionine residues at BuXI reactive site, which are involved in the inhibition, since the oxidized protein no longer inhibits factor Xa but maintains the trypsin inhibition. Quenched fluorescent substrates based on the reactive site sequence of the inhibitors were synthesized and the kinetic parameters of the hydrolysis were determined using factor Xa and trypsin. The catalytic efficiency k(cat)/K(m) 4.3 x 10(7) M(-1)sec(>-1) for Abz-VMIAALPRTMFIQ-EDDnp (lead peptide) hydrolysis by factor Xa was 10(4)-fold higher than that of Boc-Ile-Glu-Gly-Arg-AMC, widely used as factor Xa substrate. Lengthening of the substrate changed its susceptibility to factor Xa hydrolysis. Both methionine residues in the substrate influence the binding to factor Xa. Serine replacement of threonine (P(1)') decreases the catalytic efficiency by four orders of magnitude. Factor Xa did not hydrolyze the substrate containing the reactive site sequence of BvTI, that inhibits trypsin inhibitor but not factor Xa. Abz-VMIAALPRTMFIQ-EDDnp prolonged both the prothrombin time and the activated partial thromboplastin time, and the other modified substrates used in this experiment altered blood-clotting assays.

  9. Role of Factor Xa Inhibitors in Cancer-Associated Thrombosis: Any New Data?

    PubMed Central

    Zalpour, Ali; Kroll, Michael H.; Afshar-Kharghan, Vahid; Yusuf, Syed Wamique; Escalante, Carmen

    2011-01-01

    The association between cancer and venous thromboembolism (VTE) has been well documented in the literature. Prevention and treatment of VTE in cancer patients is imperative. Typically, the mainstay regimen for VTE prevention and treatment has been anticoagulation therapy, unless contraindicated. This therapy consists of unfractionated heparin (UFH), low-molecular-weight heparin (LMWH), factor Xa inhibitor, or vitamin K antagonist (VKA). Current guidelines recommend LMWH over VKA for the treatment of VTE in cancer patients. Factor-specific anticoagulants have been proven safe and effective, and recently factor Xa inhibitors have emerged as a treatment alternative to heparins and VKA. Currently, three factor Xa inhibitors have been identified: fondaparinux (the only one approved so far by the US Food and Drug Administration), idraparinux (in clinical trials), and idrabiotaparinux (in clinical trials). This paper will examine the role of these agents, focusing on fondaparinux, for the prevention and treatment of VTE in cancer patients. PMID:22013445

  10. Venous Thromboembolism Prophylaxis After TKA: Aspirin, Warfarin, Enoxaparin, or Factor Xa Inhibitors?

    PubMed

    Bala, Abiram; Huddleston, James I; Goodman, Stuart B; Maloney, William J; Amanatullah, Derek F

    2017-09-01

    There is considerable debate regarding the ideal agent for venous thromboembolism (VTE) prophylaxis after TKA. Numerous studies and meta-analyses have yet to provide a clear answer and often omit one or more of the commonly used agents such as aspirin, warfarin, enoxaparin, and factor Xa inhibitors. Using a large database analysis, we asked: (1) What are the differences in VTE incidence in primary TKA after administration of aspirin, warfarin, enoxaparin, or factor Xa inhibitors? (2) What are the differences in bleeding risk among these four agents? (3) How has use of these agents changed with time? We queried a combined Humana and Medicare database between 2007 and Quarter 1 of 2016, and identified all primary TKAs performed using ICD-9 and Current Procedural Terminology codes. All patients who had any form of antiplatelet or anticoagulation prescribed within 1 year before TKA were excluded from our study cohort. We then identified patients who had either aspirin, warfarin, enoxaparin, or factor Xa inhibitors prescribed within 2 weeks of primary TKA. Each cohort was matched by age and sex. Elixhauser comorbidities and Charlson Comorbidity Index for each group were calculated. We identified 1016 patients with aspirin, and age- and sex-matched 6096 patients with enoxaparin, 6096 patients with warfarin, and 5080 patients with factor Xa inhibitors. Using ICD-9 codes, with the understanding that patients at greater risk may have had more-attentive surveillance, the incidence of postoperative deep venous thrombosis (DVT), pulmonary embolism (PE), bleeding-related complications (bleeding requiring surgical intervention, hemorrhage, hematoma, hemarthrosis), postoperative anemia, and transfusion were identified at 2 weeks, 30 days, 6 weeks, and 90 days postoperatively. A four-way chi-squared test was used to determine statistical significance. Utilization was calculated using compound annual growth rate. There was a difference in the incidence of DVT at 90 days (p < 0

  11. Kinetic characterization of the protein Z-dependent protease inhibitor reaction with blood coagulation factor Xa.

    PubMed

    Huang, Xin; Swanson, Richard; Broze, George J; Olson, Steven T

    2008-10-31

    Protein Z-dependent protease inhibitor (ZPI) is a recently identified member of the serpin superfamily that functions as a cofactor-dependent regulator of blood coagulation factors Xa (FXa) and XIa. Here we show that ZPI and its cofactor, protein Z (PZ), inhibit procoagulant membrane-bound factor Xa by the branched pathway acyl-intermediate trapping mechanism used by other serpins, but with significant variations of this mechanism that are unique to ZPI. Rapid kinetic analyses showed that the reaction proceeded by the initial assembly of a membrane-associated PZ-ZPI-FXa Michaelis complex (K(M) 53+/-5 nM) followed by conversion to a stable ZPI-FXa complex (k(lim) 1.2+/-0.1 s(-1)). Cofactor premixing experiments together with independent kinetic analyses of ZPI-PZ and factor Xa-PZ-membrane complex formation suggested that assembly of the Michaelis complex through either ZPI-PZ-lipid or factor Xa-PZ-lipid intermediates was rate-limiting. Reaction stoichiometry analyses and native PAGE showed that for every factor Xa molecule inhibited by ZPI, two serpin molecules were cleaved. Native PAGE and immunoblotting showed that PZ dissociated from ZPI once ZPI forms a stable complex with FXa, and kinetic analyses confirmed that PZ acted catalytically to accelerate the membrane-dependent ZPI-factor Xa reaction. The ZPI-FXa complex was only transiently stable and dissociated with a rate constant that showed a bell-shaped pH dependence indicative of participation of factor Xa active-site residues. The complex was detectable by SDS-PAGE when denatured at low pH, consistent with it being a kinetically trapped covalent acyl-intermediate. Together our findings show that ZPI functions like other serpins to regulate the activity of FXa but in a manner uniquely dependent on protein Z, procoagulant membranes, and pH.

  12. Structure and property based design of factor Xa inhibitors: biaryl pyrrolidin-2-ones incorporating basic heterocyclic motifs.

    PubMed

    Young, Robert J; Borthwick, Alan D; Brown, David; Burns-Kurtis, Cynthia L; Campbell, Matthew; Chan, Chuen; Charbaut, Marie; Convery, Máire A; Diallo, Hawa; Hortense, Eric; Irving, Wendy R; Kelly, Henry A; King, N Paul; Kleanthous, Savvas; Mason, Andrew M; Pateman, Anthony J; Patikis, Angela N; Pinto, Ivan L; Pollard, Derek R; Senger, Stefan; Shah, Gita P; Toomey, John R; Watson, Nigel S; Weston, Helen E; Zhou, Ping

    2008-01-01

    Structure and property based drug design was exploited in the synthesis of sulfonamidopyrrolidin-2-one-based factor Xa (fXa) inhibitors, incorporating basic biaryl P4 groups, producing highly potent inhibitors with significant anticoagulant activities and encouraging oral pharmacokinetic profiles.

  13. Structure and property based design of factor Xa inhibitors: pyrrolidin-2-ones with biaryl P4 motifs.

    PubMed

    Young, Robert J; Borthwick, Alan D; Brown, David; Burns-Kurtis, Cynthia L; Campbell, Matthew; Chan, Chuen; Charbaut, Marie; Chung, Chun-wa; Convery, Máire A; Kelly, Henry A; Paul King, N; Kleanthous, Savvas; Mason, Andrew M; Pateman, Anthony J; Patikis, Angela N; Pinto, Ivan L; Pollard, Derek R; Senger, Stefan; Shah, Gita P; Toomey, John R; Watson, Nigel S; Weston, Helen E

    2008-01-01

    Structure and property based drug design was exploited in the synthesis of sulfonamidopyrrolidin-2-one-based factor Xa (fXa) inhibitors, incorporating biaryl P4 groups, producing highly potent inhibitors with encouraging oral pharmacokinetic profiles and significant but sub-optimal anticoagulant activities.

  14. Pharmacophore identification, in silico screening, and virtual library design for inhibitors of the human factor Xa.

    PubMed

    Krovat, Eva M; Frühwirth, Karin H; Langer, Thierry

    2005-01-01

    Factor Xa inhibitors are innovative anticoagulant agents that provide a better safety/efficacy profile compared to other anticoagulative drugs. A chemical feature-based modeling approach was applied to identify crucial pharmacophore patterns from 3D crystal structures of inhibitors bound to human factor Xa (Pdb entries 1fjs, 1kns, 1eqz) using the software LIGANDSCOUT and CATALYST. The complex structures were selected regarding the criteria of high inhibitory potency (i.e. all ligands show K(i) values against factor Xa in the subnanomolar range) and good resolution (i.e. at least 2.2 A) in order to generate selective and high quality pharmacophore models. The resulting chemical-feature based hypotheses were used for virtual screening of commercial molecular databases such as the WDI database. Furthermore, a ligand-based molecular modeling approach was performed to obtain common-feature hypotheses that represent the relevant chemical interactions between 10 bioactive factor Xa inhibitors and the protein, respectively. In a next step a virtual combinatorial library was designed in order to generate new compounds with similar chemical and spatial properties as known inhibitors. The software tool ILIB DIVERSE was used for this procedure in order to provide new scaffolds of this group of anticoagulants. Finally we present the combination of these two techniques, hence virtual screening was performed with selective pharmacophore models in a focused virtual combinatorial database. De novo derived molecular scaffolds that were able to adequately satisfy the pharmacophore criteria are revealed and are promising templates for candidates for further development.

  15. Chromogenic laboratory assays to measure the factor Xa-inhibiting properties of apixaban--an oral, direct and selective factor Xa inhibitor.

    PubMed

    Becker, Richard C; Yang, Hongqiu; Barrett, Yuchen; Mohan, Puneet; Wang, Jessie; Wallentin, Lars; Alexander, John H

    2011-08-01

    An ability to readily determine an anticoagulant effect with an emerging class of direct, active site, oral factor Xa inhibitors is viewed by the medical community as attractive and by some as an absolute requirement for their use in clinical practice. We performed a pharmacokinetic and pharmacodynamic substudy in APPRAISE-1-a study of apixaban in patients with acute coronary syndrome(ACS). A total of 1691 patients had blood sampled for apixaban plasma concentrations using mass spectrometry/high performance liquid chromatography and anti-Xa activity using a chromogenic assay employing either low molecular weight heparin or apixaban as reference standards. Anti-Xa activity, determined by either anti-Xa-LMWH (r = 0.9671; P < 0.0001) or anti-Xa-apixaban (r = 0.9669; P < 0.0001) correlated strongly and in a linear fashion with apixaban plasma concentrations. The correlations for each method were equally strong at low (<100 ng/ml) (r = 0.86, P < 0.0001; r = 0.85, P < 0.0001), intermediate(100-200 ng/ml) (r = 0.73, P < 0.0001; r = 0.69, P < 0.0001) and high (>200 ng/ml) (r = 0.91, P < 0.0001; r = 0.91, P < 0.0001) plasma concentrations of apixaban, respectively. Our pharmacokinetic and pharmacodynamic substudy suggests that an apixaban-mediated anticoagulant effect can be detected even at very low plasma concentrations using a standard laboratory chromogenic anti-Xa assay with either LMWH or apixaban calibrators. While establishing parameters for safety and efficacy will require further investigation, an ability to discern the presence of a drug effect may provide clinically useful information.

  16. New advances in the discovery of thrombin and factor Xa inhibitors.

    PubMed

    Vacca, J P

    2000-08-01

    The search for the ideal anticoagulant has spanned decades and has resulted in several strategies including the clinical use of heparin, low molecular weight heparins, and the vitamin K antagonist warfarin. Over the past five years, many groups have reported preclinical results with direct-acting thrombin inhibitors and several of these are now moving into clinical trials. In addition, many groups have disclosed the discovery of potent, orally bioavailable factor Xa inhibitors. Several of these compounds are now in early clinical trials and the results are forthcoming.

  17. Selective and dual action orally active inhibitors of thrombin and factor Xa.

    PubMed

    Young, Robert J; Brown, David; Burns-Kurtis, Cynthia L; Chan, Chuen; Convery, Máire A; Hubbard, Julia A; Kelly, Henry A; Pateman, Anthony J; Patikis, Angela; Senger, Stefan; Shah, Gita P; Toomey, John R; Watson, Nigel S; Zhou, Ping

    2007-05-15

    The synthetic entry to new classes of dual fXa/thrombin and selective thrombin inhibitors with significant oral bioavailability is described. This was achieved through minor modifications to the sulfonamide group in our potent and selective fXa inhibitor (E)-2-(5-chlorothien-2-yl)-N-{(3S)-1-[(1S)-1-methyl-2-(morpholin-4-yl)-2-oxoethyl]-2-oxopyrrolidin-3-yl}ethenesulfonamide and these observed activity changes have been rationalised using structural studies.

  18. Recent advances in the discovery and development of direct coagulation factor Xa inhibitors.

    PubMed

    Gould, W R; Leadley, R J

    2003-01-01

    Coronary heart disease (CHD) is the leading cause of mortality and morbidity in the United States. Currently, there are approximately 12 million Americans with CHD, which is most frequently caused by atherosclerosis. The thrombotic complications of atherosclerosis, such as acute coronary syndrome and ischemic stroke, can be fatal and those who survive such events have a far greater risk of future cardiovascular events. This huge medical need cries out for improved novel anticoagulants, antiplatelet agents, and profibrinolytic agents. These agents will successfully respond to the medical need by providing safe, effective, and easily administered treatments that have little, if any, drug and food interactions and that require minimal monitoring. The currently approved antiplatelet agent, clopidogrel, has satisfied some of these requirements and has played a large role in expanding the antithrombotic market over the past few years. New antithrombotics approaching the marketplace, such as the prodrug thrombin inhibitor ximelagatran, have promise in expanding the antithrombotic market further. Over the past two decades, the pharmaceutical industry has mounted a huge effort to develop antithrombotics that function by inhibiting key enzymes positioned at "higher" levels of the coagulation system. Direct inhibitors of factor Xa, which may provide a better safety and efficacy profile than currently available agents, appear to be the next major class of antithrombotic agents poised to take the pharmaceutical industry one step closer to delivering the ideal antithrombotic agent. This review focuses on recent innovations in the discovery and development of potent parenteral and oral direct factor Xa inhibitors.

  19. Anticoagulation beyond direct thrombin and factor Xa inhibitors: indications for targeting the intrinsic pathway?

    PubMed

    van Montfoort, Maurits L; Meijers, Joost C M

    2013-08-01

    Antithrombotic drugs like vitamin K antagonists and heparin have been the gold standard for the treatment and prevention of thromboembolic disease for many years. Unfortunately, there are several disadvantages of these antithrombotic drugs: they are accompanied by serious bleeding problems, it is necessary to monitor the therapeutic window, and there are various interactions with food and other drugs. This has led to the development of new oral anticoagulants, specifically inhibiting either thrombin or factor Xa. In terms of effectiveness, these drugs are comparable to the currently available anticoagulants; however, they are still associated with issues such as bleeding, reversal of the drug and complicated laboratory monitoring. Vitamin K antagonists, heparin, direct thrombin and factor Xa inhibitors have in common that they target key proteins of the haemostatic system. In an attempt to overcome these difficulties we investigated whether the intrinsic coagulation factors (VIII, IX, XI, XII, prekallikrein and high-molecular-weight kininogen) are superior targets for anticoagulation. We analysed epidemiological data concerning thrombosis and bleeding in patients deficient in one of the intrinsic pathway proteins. Furthermore, we discuss several thrombotic models in intrinsic coagulation factor-deficient animals. The combined results suggest that intrinsic coagulation factors could be suitable targets for anticoagulant drugs.

  20. Structure-based drug design of pyrrolidine-1, 2-dicarboxamides as a novel series of orally bioavailable factor Xa inhibitors.

    PubMed

    Van Huis, Chad A; Bigge, Christopher F; Casimiro-Garcia, Agustin; Cody, Wayne L; Dudley, Danette A; Filipski, Kevin J; Heemstra, Ronald J; Kohrt, Jeffrey T; Narasimhan, Lakshmi S; Schaum, Robert P; Zhang, Erli; Bryant, John W; Haarer, Staci; Janiczek, Nancy; Leadley, Robert J; McClanahan, Thomas; Thomas Peterson, J; Welch, Kathleen M; Edmunds, Jeremy J

    2007-06-01

    A novel series of pyrrolidine-1,2-dicarboxamides was discovered as factor Xa inhibitors using structure-based drug design. This series consisted of a neutral 4-chlorophenylurea P1, a biphenylsulfonamide P4 and a D-proline scaffold (1, IC(50) = 18 nM). Optimization of the initial hit resulted in an orally bioavailable, subnanomolar inhibitor of factor Xa (13, IC(50) = 0.38 nM), which was shown to be efficacious in a canine electrolytic model of thrombosis with minimal bleeding.

  1. A NMR and MD study of the active site of factor Xa by selective inhibitors

    NASA Astrophysics Data System (ADS)

    Doan, B. T.; Fraternali, F.; Do, Q. T.; Atkinson, R. A.; Palmas, P.; Sklenar, V.; Wildgoose, P.; Strop, P.; Saudek, V.

    1998-02-01

    The structure of two selective inhibitors obtained by the screening of a vast combinatorial library, Ac-Tyr-Ile-Arg-Ile-NH2 and Ac-(4-amino-Phe)-(Cyc.-Gly)-NH2, in the active site of the blood clotting enzyme factor Xa was determined using transferred NOE NMR and simulated annealing (SA) under NMR constraints. The refined structures of the inhibitors were docked in the active site and SA was performed inside the enzyme which has been kept as a rigid charged template. The final structures were optimised by molecular dynamics simulation of the complexes in water. The inhibitors assume a compact, very well defined conformation embedded in the binding site without blocking the catalysis. The model allows to explain the mode of action, affinity and specificity. L'étude structurale d'inhibiteurs du facteur Xa, une enzyme de coagulation, obtenus par chimie combinatoire : Ac-Tyr-Ile-Arg-Ile-NH2, Ac-(4-amino-Phe)-(Cyc.-Gly)-NH2, a été réalisée par RMN NOE de transfert et modélisation moléculaire. Les structures ont été calculées sous contraintes RMN : géométrie de distance, recuit simulé et minimisation, affinées par une recherche conformationnelle et recuit de l'inhibiteur placé dans le site actif et optimisées par simulation de dynamique moléculaire du complexe dans l'eau. L'inhibiteur présente une structure compacte positionnée dans le site d'interaction hors d'accès du site catalytique. Ce modèle permet d'expliquer le mode d'action, l'affinité et la spécificité des peptides.

  2. Parenteral administration of factor Xa/IIa inhibitors limits experimental aortic aneurysm and atherosclerosis.

    PubMed

    Moran, Corey S; Seto, Sai-Wang; Krishna, Smriti M; Sharma, Surabhi; Jose, Roby J; Biros, Erik; Wang, Yutang; Morton, Susan K; Golledge, Jonathan

    2017-02-21

    Intraluminal thrombus is a consistent feature of human abdominal aortic aneurysm (AAA). Coagulation factor Xa (FXa) catalyses FII to thrombin (FIIa). We examined the effect of FXa/FIIa inhibition on experimental aortic aneurysm in apolipoprotein E-deficient (ApoE(-/-)) mice infused with angiotensin II (AngII). The concentration of FXa within the supra-renal aorta (SRA) correlated positively with SRA diameter. Parenteral administration of enoxaparin (FXa/IIa inhibitor) and fondaparinux (FXa inhibitor) over 14 days reduced to severity of aortic aneurysm and atherosclerosis in AngII-infused ApoE(-/-) mice. Enteral administration of the FIIa inhibitor dabigatran had no significant effect. Aortic protease-activated receptor (PAR)-2 expression increased in response to AngII infusion. Fondaparinux reduced SRA levels of FXa, FIIa, PAR-2, matrix metalloproteinase (MMP)2, Smad2/3 phosphorylation, and MOMA-2 positive cells in the mouse model. FXa stimulated Smad2/3 phosphorylation and MMP2 expression in aortic vascular smooth muscle cells (VSMC) in vitro. Expression of MMP2 in FXa-stimulated VSMC was downregulated in the presence of a PAR-2 but not a PAR-1 inhibitor. These findings suggest that FXa/FIIa inhibition limits aortic aneurysm and atherosclerosis severity due to down-regulation of vascular PAR-2-mediated Smad2/3 signalling and MMP2 expression. Inhibition of FXa/FIIa may be a potential therapy for limiting aortic aneurysm.

  3. Parenteral administration of factor Xa/IIa inhibitors limits experimental aortic aneurysm and atherosclerosis

    PubMed Central

    Moran, Corey S.; Seto, Sai-Wang; Krishna, Smriti M.; Sharma, Surabhi; Jose, Roby J.; Biros, Erik; Wang, Yutang; Morton, Susan K.; Golledge, Jonathan

    2017-01-01

    Intraluminal thrombus is a consistent feature of human abdominal aortic aneurysm (AAA). Coagulation factor Xa (FXa) catalyses FII to thrombin (FIIa). We examined the effect of FXa/FIIa inhibition on experimental aortic aneurysm in apolipoprotein E-deficient (ApoE−/−) mice infused with angiotensin II (AngII). The concentration of FXa within the supra-renal aorta (SRA) correlated positively with SRA diameter. Parenteral administration of enoxaparin (FXa/IIa inhibitor) and fondaparinux (FXa inhibitor) over 14 days reduced to severity of aortic aneurysm and atherosclerosis in AngII-infused ApoE−/− mice. Enteral administration of the FIIa inhibitor dabigatran had no significant effect. Aortic protease-activated receptor (PAR)-2 expression increased in response to AngII infusion. Fondaparinux reduced SRA levels of FXa, FIIa, PAR-2, matrix metalloproteinase (MMP)2, Smad2/3 phosphorylation, and MOMA-2 positive cells in the mouse model. FXa stimulated Smad2/3 phosphorylation and MMP2 expression in aortic vascular smooth muscle cells (VSMC) in vitro. Expression of MMP2 in FXa-stimulated VSMC was downregulated in the presence of a PAR-2 but not a PAR-1 inhibitor. These findings suggest that FXa/FIIa inhibition limits aortic aneurysm and atherosclerosis severity due to down-regulation of vascular PAR-2-mediated Smad2/3 signalling and MMP2 expression. Inhibition of FXa/FIIa may be a potential therapy for limiting aortic aneurysm. PMID:28220880

  4. Structure- and property-based design of factor Xa inhibitors: pyrrolidin-2-ones with acyclic alanyl amides as P4 motifs.

    PubMed

    Young, Robert J; Campbell, Matthew; Borthwick, Alan D; Brown, David; Burns-Kurtis, Cynthia L; Chan, Chuen; Convery, Máire A; Crowe, Miriam C; Dayal, Satish; Diallo, Hawa; Kelly, Henry A; King, N Paul; Kleanthous, Savvas; Mason, Andrew M; Mordaunt, Jackie E; Patel, Champa; Pateman, Anthony J; Senger, Stefan; Shah, Gita P; Smith, Paul W; Watson, Nigel S; Weston, Helen E; Zhou, Ping

    2006-12-01

    Structure-based drug design was exploited in the synthesis of 3-(6-chloronaphth-2-ylsulfonyl)aminopyrrolidin-2-one-based factor Xa (fXa) inhibitors, incorporating an alanylamide P4 group with acyclic tertiary amide termini. Optimized hydrophobic contacts of one amide substituent in P4 were complemented by hydrophobicity-modulating features in the second, producing potent fXa inhibitors including examples with excellent anticoagulant properties.

  5. The discovery of potent and long-acting oral factor Xa inhibitors with tetrahydroisoquinoline and benzazepine P4 motifs.

    PubMed

    Watson, Nigel S; Adams, Carl; Belton, David; Brown, David; Burns-Kurtis, Cynthia L; Chaudry, Laiq; Chan, Chuen; Convery, Máire A; Davies, David E; Exall, Anne M; Harling, John D; Irvine, Stephanie; Irving, Wendy R; Kleanthous, Savvas; McLay, Iain M; Pateman, Anthony J; Patikis, Angela N; Roethke, Theresa J; Senger, Stefan; Stelman, Gary J; Toomey, John R; West, Robert I; Whittaker, Caroline; Zhou, Ping; Young, Robert J

    2011-03-15

    The discovery and evaluation of potent and long-acting oral sulfonamidopyrrolidin-2-one factor Xa inhibitors with tetrahydroisoquinoline and benzazepine P4 motifs are described. Unexpected selectivity issues versus tissue plasminogen activator in the former series were addressed in the later, delivering a robust candidate for progression towards clinical studies. Copyright © 2011 Elsevier Ltd. All rights reserved.

  6. Basis for the Specificity and Activation of the Serpin Protein Z-dependent Proteinase Inhibitor (ZPI) as an Inhibitor of Membrane-associated Factor Xa

    SciTech Connect

    Huang, Xin; Dementiev, Alexey; Olson, Steven T.; Gettins, Peter G.W.

    2012-12-13

    The serpin ZPI is a protein Z (PZ)-dependent specific inhibitor of membrane-associated factor Xa (fXa) despite having an unfavorable P1 Tyr. PZ accelerates the inhibition reaction {approx}2000-fold in the presence of phospholipid and Ca{sup 2+}. To elucidate the role of PZ, we determined the x-ray structure of Gla-domainless PZ (PZ{sub {Delta}GD}) complexed with protein Z-dependent proteinase inhibitor (ZPI). The PZ pseudocatalytic domain bound ZPI at a novel site through ionic and polar interactions. Mutation of four ZPI contact residues eliminated PZ binding and membrane-dependent PZ acceleration of fXa inhibition. Modeling of the ternary Michaelis complex implicated ZPI residues Glu-313 and Glu-383 in fXa binding. Mutagenesis established that only Glu-313 is important, contributing {approx}5-10-fold to rate acceleration of fXa and fXIa inhibition. Limited conformational change in ZPI resulted from PZ binding, which contributed only {approx}2-fold to rate enhancement. Instead, template bridging from membrane association, together with previously demonstrated interaction of the fXa and ZPI Gla domains, resulted in an additional {approx}1000-fold rate enhancement. To understand why ZPI has P1 tyrosine, we examined a P1 Arg variant. This reacted at a diffusion-limited rate with fXa, even without PZ, and predominantly as substrate, reflecting both rapid acylation and deacylation. P1 tyrosine thus ensures that reaction with fXa or most other arginine-specific proteinases is insignificant unless PZ binds and localizes ZPI and fXa on the membrane, where the combined effects of Gla-Gla interaction, template bridging, and interaction of fXa with Glu-313 overcome the unfavorability of P1 Tyr and ensure a high rate of reaction as an inhibitor.

  7. Basis for the specificity and activation of the serpin protein Z-dependent proteinase inhibitor (ZPI) as an inhibitor of membrane-associated factor Xa.

    PubMed

    Huang, Xin; Dementiev, Alexey; Olson, Steven T; Gettins, Peter G W

    2010-06-25

    The serpin ZPI is a protein Z (PZ)-dependent specific inhibitor of membrane-associated factor Xa (fXa) despite having an unfavorable P1 Tyr. PZ accelerates the inhibition reaction approximately 2000-fold in the presence of phospholipid and Ca(2+). To elucidate the role of PZ, we determined the x-ray structure of Gla-domainless PZ (PZ(DeltaGD)) complexed with protein Z-dependent proteinase inhibitor (ZPI). The PZ pseudocatalytic domain bound ZPI at a novel site through ionic and polar interactions. Mutation of four ZPI contact residues eliminated PZ binding and membrane-dependent PZ acceleration of fXa inhibition. Modeling of the ternary Michaelis complex implicated ZPI residues Glu-313 and Glu-383 in fXa binding. Mutagenesis established that only Glu-313 is important, contributing approximately 5-10-fold to rate acceleration of fXa and fXIa inhibition. Limited conformational change in ZPI resulted from PZ binding, which contributed only approximately 2-fold to rate enhancement. Instead, template bridging from membrane association, together with previously demonstrated interaction of the fXa and ZPI Gla domains, resulted in an additional approximately 1000-fold rate enhancement. To understand why ZPI has P1 tyrosine, we examined a P1 Arg variant. This reacted at a diffusion-limited rate with fXa, even without PZ, and predominantly as substrate, reflecting both rapid acylation and deacylation. P1 tyrosine thus ensures that reaction with fXa or most other arginine-specific proteinases is insignificant unless PZ binds and localizes ZPI and fXa on the membrane, where the combined effects of Gla-Gla interaction, template bridging, and interaction of fXa with Glu-313 overcome the unfavorability of P1 Tyr and ensure a high rate of reaction as an inhibitor.

  8. Structure and property based design of factor Xa inhibitors: pyrrolidin-2-ones with monoaryl P4 motifs.

    PubMed

    Kleanthous, Savvas; Borthwick, Alan D; Brown, David; Burns-Kurtis, Cynthia L; Campbell, Matthew; Chaudry, Laiq; Chan, Chuen; Clarte, Marie-Olive; Convery, Máire A; Harling, John D; Hortense, Eric; Irving, Wendy R; Irvine, Stephanie; Pateman, Anthony J; Patikis, Angela N; Pinto, Ivan L; Pollard, Derek R; Roethka, Theresa J; Senger, Stefan; Shah, Gita P; Stelman, Gary J; Toomey, John R; Watson, Nigel S; West, Robert I; Whittaker, Caroline; Zhou, Ping; Young, Robert J

    2010-01-15

    Structure and property based drug design was exploited in the synthesis of sulfonamidopyrrolidin-2-one-based factor Xa inhibitors, incorporating neutral and basic monoaryl P4 groups, ultimately producing potent inhibitors with effective levels of anticoagulant activity and extended oral pharmacokinetic profiles. However, time dependant inhibition of Cytochrome P450 3A4 was a particular issue with this series. Copyright 2009 Elsevier Ltd. All rights reserved.

  9. Engineering Factor Xa Inhibitor with Multiple Platelet-Binding Sites Facilitates its Platelet Targeting

    NASA Astrophysics Data System (ADS)

    Zhu, Yuanjun; Li, Ruyi; Lin, Yuan; Shui, Mengyang; Liu, Xiaoyan; Chen, Huan; Wang, Yinye

    2016-07-01

    Targeted delivery of antithrombotic drugs centralizes the effects in the thrombosis site and reduces the hemorrhage side effects in uninjured vessels. We have recently reported that the platelet-targeting factor Xa (FXa) inhibitors, constructed by engineering one Arg-Gly-Asp (RGD) motif into Ancylostoma caninum anticoagulant peptide 5 (AcAP5), can reduce the risk of systemic bleeding than non-targeted AcAP5 in mouse arterial injury model. Increasing the number of platelet-binding sites of FXa inhibitors may facilitate their adhesion to activated platelets, and further lower the bleeding risks. For this purpose, we introduced three RGD motifs into AcAP5 to generate a variant NR4 containing three platelet-binding sites. NR4 reserved its inherent anti-FXa activity. Protein-protein docking showed that all three RGD motifs were capable of binding to platelet receptor αIIbβ3. Molecular dynamics simulation demonstrated that NR4 has more opportunities to interact with αIIbβ3 than single-RGD-containing NR3. Flow cytometry analysis and rat arterial thrombosis model further confirmed that NR4 possesses enhanced platelet targeting activity. Moreover, NR4-treated mice showed a trend toward less tail bleeding time than NR3-treated mice in carotid artery endothelium injury model. Therefore, our data suggest that engineering multiple binding sites in one recombinant protein is a useful tool to improve its platelet-targeting efficiency.

  10. Contemporary developments in the discovery of selective factor Xa inhibitors: A review.

    PubMed

    Patel, Nirav R; Patel, Dushyant V; Murumkar, Prashant R; Yadav, Mange Ram

    2016-10-04

    Thrombosis is a leading cause of death in cardiovascular diseases such as myocardial infarction (MI), unstable angina and acute coronary syndrome (ACS) in the industrialized world. Venous thromboembolism is observed in about 1 million people every year in United States causing significant morbidity and mortality. Conventional antithrombotic therapy has been reported to have several disadvantages and limitations like inconvenience in oral administration, bleeding risks (heparin analogs), narrow therapeutic window and undesirable interactions with food and drugs (vitamin K antagonist-warfarin). The unmet medical demand for orally active safe anticoagulants has generated widespread interest among the medicinal chemists engaged in this field. To modulate blood coagulation, various enzymes involved in the coagulation process have received great attention as potential targets by various research groups for the development of oral anticoagulants. Among these enzymes, factor Xa (FXa) has remained the centre of attention in the last decade. Intensive research efforts have been made by various research groups for the development of small, safe and orally bioavailable FXa inhibitors. This review is an attempt to compile the research work of various researchers in the direction of development of FXa inhibitors reported since 2010 onward.

  11. Effect of famotidine on the pharmacokinetics of apixaban, an oral direct factor Xa inhibitor

    PubMed Central

    Upreti, Vijay V; Song, Yan; Wang, Jessie; Byon, Wonkyung; Boyd, Rebecca A; Pursley, Janice M; LaCreta, Frank; Frost, Charles E

    2013-01-01

    Background Apixaban is an oral, selective, direct factor Xa inhibitor approved for thromboprophylaxis after orthopedic surgery and stroke prevention in patients with atrial fibrillation, and under development for treatment of venous thromboembolism. This study investigated the effect of a gastric acid suppressant, famotidine (a histamine H2-receptor antagonist), on the pharmacokinetics of apixaban in healthy subjects. Methods This two-period, two-treatment crossover study randomized 18 healthy subjects to receive a single oral dose of apixaban 10 mg with and without a single oral dose of famotidine 40 mg administered 3 hours before dosing with apixaban. Plasma apixaban concentrations were measured up to 60 hours post-dose and pharmacokinetic parameters were calculated. Results Famotidine did not affect maximum apixaban plasma concentration (Cmax) or area under the plasma concentration-time curve from zero to infinite time (AUC∞). Point estimates for ratios of geometric means with and without famotidine were close to unity for Cmax (0.978) and AUC∞ (1.007), and 90% confidence intervals were entirely contained within the 80%–125% no-effect interval. Administration of apixaban alone and with famotidine was well tolerated. Conclusion Famotidine does not affect the pharmacokinetics of apixaban, consistent with the physicochemical properties of apixaban (lack of an ionizable group and pH-independent solubility). Apixaban pharmacokinetics would not be affected by an increase in gastrointestinal pH due to underlying conditions (eg, achlorhydria), or by gastrointestinal pH-mediated effects of other histamine H2-receptor antagonists, antacids, or proton pump inhibitors. Given that famotidine is also an inhibitor of the human organic cation transporter (hOCT), these results indicate that apixaban pharmacokinetics are not influenced by hOCT uptake transporter inhibitors. Overall, these results support that apixaban can be administered without regard to coadministration

  12. Potent Direct Inhibitors of Factor Xa Based on the Tetrahydroisoquinoline Scaffold

    PubMed Central

    Al-Horani, Rami A.; Mehta, Akul Y.; Desai, Umesh R.

    2012-01-01

    Direct inhibition of coagulation factor Xa (FXa) carries significant promise for developing effective and safe anticoagulants. Although a large number of FXa inhibitors have been studied, each can be classified as either possessing a highly flexible or a rigid core scaffold. We reasoned that an intermediate level of flexibility will provide high selectivity for FXa considering that its active site is less constrained in comparison to thrombin and more constrained as compared to trypsin. We studied several core scaffolds including 1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid for direct FXa inhibition. Using a genetic algorithm-based docking and scoring approach, a promising candidate 23 was identified, synthesized, and found to inhibit FXa with a Ki of 28 μM. Optimization of derivative 23 resulted in the design of a potent dicarboxamide 47, which displayed a Ki of 135 nM. Dicarboxamide 47 displayed at least 1852-fold selectivity for FXa inhibition over other coagulation enzymes and doubled PT and aPTT of human plasma at 17.1 μM and 20.2 μM, respectively, which are comparable to those of clinically relevant agents. Dicarboxamide 47 is expected to serve as an excellent lead for further anticoagulant discovery. PMID:22770607

  13. Factor Xa inhibitors versus vitamin K antagonists for preventing cerebral or systemic embolism in patients with atrial fibrillation.

    PubMed

    Bruins Slot, Karsten M H; Berge, Eivind

    2013-08-08

    Anticoagulant treatment with vitamin K antagonists (VKAs) is aimed at preventing thromboembolic complications and has been the therapy of choice for most people with non-valvular atrial fibrillation (AF) for many decades. A new class of anticoagulants, the factor Xa inhibitors, appear to have several pharmacological and practical advantages over VKAs. To assess the effectiveness and safety of treatment with factor Xa inhibitors versus VKAs for the prevention of cerebral or systemic embolic events in people with AF. We searched the trials registers of the Cochrane Stroke Group and the Cochrane Heart Group (June 2012), the Cochrane Central Register of Controlled Trials (CENTRAL) (The Cochrane Library 2012, Issue 10), MEDLINE (1950 to April 2013) and EMBASE (1980 to April 2013). In an effort to identify further published, unpublished and ongoing trials we searched trials registers and Google Scholar (July 2012). We also screened reference lists and contacted pharmaceutical companies, authors and sponsors of relevant published trials. Randomised controlled trials that directly compared the effects of long-term treatment (more than four weeks) with factor Xa inhibitors and VKAs for the prevention of cerebral and systemic embolism in patients with AF. We included patients with and without a previous stroke or TIA. The primary efficacy outcome was the composite endpoint of all strokes and other systemic embolic events. Two authors independently assessed trial quality and the risk of bias, and extracted data. We calculated a weighted estimate of the typical treatment effect across trials using the odds ratio (OR) with 95% confidence interval (CI) by means of a fixed-effect model. However, in the case of moderate or high heterogeneity of treatment effects, we used a random-effects model to compare the overall treatment effects and performed a pre-specified sensitivity analysis excluding any fully open-label studies. We included data from 42,084 participants randomised into

  14. Structure and property based design of factor Xa inhibitors: pyrrolidin-2-ones with aminoindane and phenylpyrrolidine P4 motifs.

    PubMed

    Young, Robert J; Adams, Carl; Blows, Mike; Brown, David; Burns-Kurtis, Cynthia L; Chan, Chuen; Chaudry, Laiq; Convery, Máire A; Davies, David E; Exall, Anne M; Foster, Graham; Harling, John D; Hortense, Eric; Irvine, Stephanie; Irving, Wendy R; Jackson, Steve; Kleanthous, Savvas; Pateman, Anthony J; Patikis, Angela N; Roethka, Theresa J; Senger, Stefan; Stelman, Gary J; Toomey, John R; West, Robert I; Whittaker, Caroline; Zhou, Ping; Watson, Nigel S

    2011-03-15

    The rational design, syntheses and evaluation of potent sulfonamidopyrrolidin-2-one-based factor Xa inhibitors incorporating aminoindane and phenylpyrrolidine P4 motifs are described. These series delivered highly potent anticoagulant compounds with excellent oral pharmacokinetic profiles; however, significant time dependant P450 inhibition was an issue for the aminoindane series, but this was not observed with the phenylpyrrolidine motif, which produced candidate quality molecules with potential for once-daily oral dosing in humans. Copyright © 2011 Elsevier Ltd. All rights reserved.

  15. Antithrombotic potential of GW813893: a novel, orally active, active-site directed factor Xa inhibitor.

    PubMed

    Abboud, Melanie A; Needle, Saul J; Burns-Kurtis, Cynthia L; Valocik, Richard E; Koster, Paul F; Amour, Augustin J; Chan, Chuen; Brown, David; Chaudry, Laiq; Zhou, Ping; Patikis, Angela; Patel, Champa; Pateman, Anthony J; Young, Rob J; Watson, Nigel S; Toomey, John R

    2008-07-01

    Factor Xa (FXa) has been a target of considerable interest for drug development efforts aimed at suppressing thrombosis. In this report, a new orally active, small molecule, active-site directed FXa inhibitor, GW813893, has been profiled in a succession of in vitro and in vivo assays involved in its preclinical characterization as a potential antithrombotic therapeutic. In vitro profiling of GW813893 consisted of assessing its inhibitory potential against FXa and a broad panel of related and unrelated enzymes and receptors. Additionally, the FXa inhibition potential of GW813893 was assessed in prothrombinase and plasma-based clotting assays. In vivo characterization of GW813893 consisted of thrombosis studies in a rat inferior vena cava model, a rat carotid artery thrombosis model, and a rabbit jugular thrombosis model. Bleeding studies were conducted in a rat tail transection model. Ex vivo determinations of compound effects on FX and clotting activity were also undertaken. GW813893 was more than 90-fold selective over all enzymes tested, and it inhibited FXa and prothrombinase activity with a Ki of 4.0 nM and 9.7 nM, respectively. In vivo, GW813893 concentration-dependently suppressed thrombotic activity in all models tested. The antithrombotic activity correlated with the suppression of plasma-based clotting activity and the inhibition of plasma FX activity (P < 0.02). Over the antithrombotic dose-range, an increased bleeding diathesis was not observed. These experiments demonstrate that GW813893 is a potent, selective, orally active inhibitor of FXa. The data suggest that GW813893 has robust antithrombotic potential at doses that have no detectable hemostasis liability. Collectively, the profile suggests that GW813893 has the preclinical pharmacology underpinnings of an oral antithrombotic therapeutic.

  16. The direct factor Xa inhibitor Rivaroxaban reduces platelet activation in congestive heart failure.

    PubMed

    Flierl, Ulrike; Fraccarollo, Daniela; Micka, Jan; Bauersachs, Johann; Schäfer, Andreas

    2013-08-01

    Platelet activation in congestive heart failure (CHF) contributes to an increased risk for thromboembolic complications. Rivaroxaban, the first oral direct FXa inhibitor is approved in Europe for prevention and treatment of venous thrombosis, pulmonary embolism, and prevention of thromboembolic events in atrial fibrillation. As heart failure is an important risk factor for thromboembolism and increased platelet activation is common in heart failure, we investigated the potential effect of Rivaroxaban treatment on platelets in an experimental CHF model. Chronic myocardial infarction was induced in male Wistar rats by coronary ligation. Rats were randomized to placebo or Rivaroxaban (3 and 10mg/kg once daily). After 10 weeks platelet activation was assessed. Platelet-bound fibrinogen, detected by flow-cytometry, was significantly increased in CHF-Placebo (p<0.05) and reduced following treatment with Rivaroxaban (p<0.05 vs. CHF-Placebo). ADP-induced aggregation was significantly enhanced in CHF-Placebo vs. sham-operated animals (p<0.05) and normalized following chronic FXa inhibition (p<0.05 vs. CHF-Placebo). In separate in vitro experiments, attenuated platelet aggregation was present after incubating whole blood directly with Rivaroxaban but absent when the experiment was performed in platelet-rich plasma only. Thus, a direct effect on platelets could be excluded. Chronic direct factor Xa inhibition using Rivaroxaban reduces platelet activation in CHF rats by attenuating the secondary phase of ADP-induced platelet aggregation. Thus, Rivaroxaban may constitute a useful approach to prevent thromboembolic complications and reduce platelet activation in CHF at the same time. Copyright © 2013 Elsevier Ltd. All rights reserved.

  17. Structure-guided creation of AcAP5-derived and platelet targeted factor Xa inhibitors.

    PubMed

    Zhu, Yuanjun; Lin, Yuan; Liu, Aihua; Shui, Mengyang; Li, Ruyi; Liu, Xiaoyan; Hu, Wenhui; Wang, Yinye

    2015-06-15

    Anticoagulants and anti-platelet agents are simultaneously administrated in clinical practice (i.e. percutaneous coronary intervention), which cause significant risk of systemic bleeding. Targeted delivery of anticoagulants to the activated platelets at sites of vascular injuries may condense the site-specific anticoagulant effect and reduce the hemorrhage side effects in uninjured vessels. To this end, we prepared three ancylostoma caninum anticoagulant peptide 5 (AcAP5) variants NR1, NR2 and NR3 engineered with a platelet-binding Arg-Gly-Asp (RGD) motif and evaluated their anti-Factor Xa (FXa) and platelet-binding effects. These RGD-containing AcAP5 variants were capable of interacting with platelet receptor αIIbβ3 as shown in computational analysis. All variants, especially NR2 and NR3, retained entirely the anti-FXa function of parent AcAP5. Moreover, they prevented the formation of occlusive thrombi in rat carotid artery injury model, suggesting that they inhibit platelet aggregation in vivo. Further functional investigation of NR3 demonstrated that NR3 inhibited platelet aggregation in vitro and FXa activity in vivo, and prolonged the coagulation time, all in a dose-dependent manner. Through flow cytometry assay, we confirmed the binding of NR3 to αIIbβ3 receptor. In mouse model of carotid artery endothelium injury, NR3-treated mice showed less tail bleeding time than AcAP5-treated mice, and aspirin plus NR3 treatment exhibited moderate reduction of blood loss compared with aspirin plus AcAP5 treatment. These results indicate the feasibility to engineer a novel FXa inhibitor specifically targeting the activated platelets, which centralizes its anticoagulation efficacy in the injured vascular endothelium and reduces the risk of systemic bleeding. Copyright © 2015 Elsevier Inc. All rights reserved.

  18. Discovery of glycyrrhetinic acid as an orally active, direct inhibitor of blood coagulation factor xa.

    PubMed

    Jiang, Lilong; Wang, Qiong; Shen, Shu; Xiao, Tongshu; Li, Youbin

    2014-03-01

    Factor Xa (FXa) plays an important role in blood coagulation. This study investigated glycyrrhetinic acid, a small molecule derived from Chinese herbs, and whether it has a direct inhibitory effect on FXa to display its anticoagulant activity. Enzyme activities of FXa, plasmin, trypsin and thrombin, inhibition of FXa enzyme kinetics and plasma clotting time by glycyrrhentinic acid were performed in vitro. A rat tail-bleeding model and a rat venous stasis model were also used to evaluate in vivo tail-bleeding time and thrombus formation, respectively. Glycyrrhetinic acid in vitro directly inhibited FXa uncompetitivly with IC50 of 32.6 ± 1.24 μmol/L, and displayed 2-, 14- and 20-fold selectivity for FXa when compared to plasmin, thrombin and trypsin, respectively. The plasma clotting time was increased in a dose-dependent manner. The prothrombin time doubled (PT2), when the concentration of glycyrrhetinic acid reached 2.02 mmol/L. During in vivo experiments intragastric administration of glycyrrhetinic acid caused a dose-dependent reduction in thrombus weight on the rat venous stasis model (all P<0.05). 50 mg/kg glycyrrhetinic acid resulted in 34.8% of venous thrombus weight lost, compared to the control. In addition, 200, 300 and 400 mg/kg doses of glycyrrhetinic acid caused a moderate hemorrhagic effect in the rat tail-bleeding model by prolonging bleeding time 1.1-, 1.5- and 1.9-fold compared to the control, respectively. Glycyrrhetinic acid is a direct inhibitor of FXa that is effective by oral administration, and with further research could be used to treat blood coagulation disorders. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Structure-based design of novel guanidine/benzamidine mimics: potent and orally bioavailable factor Xa inhibitors as novel anticoagulants.

    PubMed

    Lam, Patrick Y S; Clark, Charles G; Li, Renhua; Pinto, Donald J P; Orwat, Michael J; Galemmo, Robert A; Fevig, John M; Teleha, Christopher A; Alexander, Richard S; Smallwood, Angela M; Rossi, Karen A; Wright, Matthew R; Bai, Stephen A; He, Kan; Luettgen, Joseph M; Wong, Pancras C; Knabb, Robert M; Wexler, Ruth R

    2003-10-09

    As part of an ongoing effort to prepare orally active factor Xa inhibitors using structure-based drug design techniques and molecular recognition principles, a systematic study has been performed on the pharmacokinetic profile resulting from replacing the benzamidine in the P1 position with less basic benzamidine mimics or neutral residues. It is demonstrated that lowering the pK(a) of the P1 ligand resulted in compounds (3-benzylamine, 15a; 1-aminoisoquinoline, 24a; 3-aminobenzisoxazole, 23a; 3-phenylcarboxamide, 22b; and 4-methoxyphenyl, 22a) with improved pharmacokinetic features mainly as a result of decreased clearance, increased volume of distribution, and enhanced oral absorption. This work resulted in a series of potent and orally bioavailable factor Xa inhibitors that ultimately led to the discovery of SQ311, 24a. SQ311, which utilizes a 1-aminoisoquinoline as the P1 ligand, inhibits factor Xa with a K(i) of 0.33 nM and demonstrates both good in vivo antithrombotic efficacy and oral bioavailability.

  20. Safety, tolerability, pharmacodynamics, and pharmacokinetics of rivaroxaban--an oral, direct factor Xa inhibitor--are not affected by aspirin.

    PubMed

    Kubitza, Dagmar; Becka, Michael; Mueck, Wolfgang; Zuehlsdorf, Michael

    2006-09-01

    Rivaroxaban (BAY 59-7939) is an oral, direct Factor Xa inhibitor in advanced clinical development for the prevention and treatment of thromboembolic disorders. This was a randomized, 2-way crossover study in healthy male subjects, with an aspirin run-in period, to examine whether aspirin influences the safety, tolerability, pharmacodynamics, and pharmacokinetics of rivaroxaban. All treatments were well tolerated; drug-related adverse events were mild and transient. Aspirin did not alter the effects of rivaroxaban on Factor Xa activity or clotting tests. Platelet aggregation and bleeding time were not affected by rivaroxaban, and rivaroxaban did not influence the effects of aspirin on these parameters to a clinically relevant extent. Aspirin did not affect the pharmacokinetics of rivaroxaban, including the fraction unbound. This study suggests that there is no clinically relevant interaction between rivaroxaban and aspirin and that the 2 drugs could be administered concomitantly at the doses used in this study.

  1. Apixaban, a direct factor Xa inhibitor, inhibits tissue-factor induced human platelet aggregation in vitro: comparison with direct inhibitors of factor VIIa, XIa and thrombin.

    PubMed

    Wong, Pancras C; Jiang, Xiaosui

    2010-08-01

    Apixaban is an oral, direct and highly selective factor Xa (FXa) inhibitor in late-stage clinical development. This study evaluated the in vitro effect of apixaban on human platelet aggregation induced by thrombin derived via the extrinsic pathway. Direct inhibitors of FXa (rivaroxaban), FVIIa (BMS-593214), thrombin (dabigatran, argatroban) and FXIa (BMS-262084) were included for comparison. Citrated human platelets-rich plasma (PRP) was treated with 50 mg/ml corn trypsin inhibitor (to block the contact factor pathway) and 3 mM H-Gly-Pro-Arg-Pro-OH-AcOH (to prevent fibrin polymerisation). Human tissue factor (TF) (Innovin; dilution 1:1,000 to 1:1,500) plus 7.5 mM CaCl2 was added to PRP pre-incubated with vehicle or increasing concentrations of inhibitors. The TF-induced platelet aggregation was measured by optical aggregometry. TF produced 85 +/- 3% aggregation of human platelets in the vehicle-treated group (n=10). Apixaban and other factor inhibitors, except the FXIa inhibitor, inhibited TF-induced platelet aggregation with IC50 (nM) values as follows: 4 +/- 1 (apixaban), 8 +/- 2 (rivaroxaban), 13 +/- 1 (BMS-593214), 46 +/- 1 (dabigatran) and 79 +/- 1 (argatroban). BMS-262084 (IC50 = 2.8 nM vs. human FXIa) had no effect on TF-induced platelet aggregation at 10 microM. These inhibitors at 10 microM had no effect on platelet aggregation induced by ADP and collagen, as expected from their mechanism of action. This study demonstrates that inhibition of thrombin generation by blocking upstream proteases (FVIIa and FXa) in the blood coagulation cascade is as effective as direct thrombin inhibition in preventing TF-induced platelet aggregation. Under these experimental conditions, a FXIa inhibitor did not prevent TF-induced platelet aggregation.

  2. A four component coupling strategy for the synthesis of D-phenylglycinamide-derived non-covalent factor Xa inhibitors.

    PubMed

    Sheehan, Scott M; Masters, John J; Wiley, Michael R; Young, Stephen C; Liebeschuetz, John W; Jones, Stuart D; Murray, Christopher W; Franciskovich, Jeffrey B; Engel, David B; Weber, Wayne W; Marimuthu, Jothirajah; Kyle, Jeffrey A; Smallwood, Jeffrey K; Farmen, Mark W; Smith, Gerald F

    2003-07-21

    A novel isonitrile derivative was synthesized and used in an Ugi four component coupling reaction to explore aryl group substitution effects on inhibition of the coagulation cascade serine protease factor Xa.

  3. Population Pharmacokinetic and Pharmacodynamic Modeling Analysis of GCC‐4401C, a Novel Direct Factor Xa Inhibitor, in Healthy Volunteers

    PubMed Central

    Choi, HY; Choi, S; Kim, YH

    2016-01-01

    GCC‐4401C, an orally active direct factor Xa inhibitor that is similar to rivaroxaban, is currently under development for venous thromboembolic disease (VTE). The purpose of this study was to characterize the pharmacokinetics (PKs) and pharmacodynamics (PDs) of GCC‐4401C by population modeling analysis and to predict proper dosage regimens compared to rivaroxaban using data from two phase I clinical studies. Plasma GCC‐4401C concentrations over time were best described by a two‐compartment linear model and body weight was associated with central volume of distribution. Relevant PD markers generally changed in a dose‐dependent manner and were described well with sigmoid, simple maximum effect, or linear models. GCC‐4401C was absorbed more rapidly than rivaroxaban. Comparisons based on simulations of PD marker changes over time suggest that 20 mg and 40 mg of GCC‐4401C administered under fasted status are comparable to 10 mg and 20 mg of rivaroxaban under fed status. PMID:27511836

  4. Microfluidic Chip-Based Online Screening Coupled to Mass Spectrometry: Identification of Inhibitors of Thrombin and Factor Xa.

    PubMed

    Iyer, Janaki Krishnamoorthy; Otvos, Reka A; Kool, Jeroen; Kini, R Manjunatha

    2016-02-01

    Thrombin and factor Xa (FXa) are critical enzymes of the blood coagulation cascade and are excellent targets of anticoagulant agents. Natural sources present an array of anticoagulants that can be developed as antithrombotic drugs. High-resolution, online screening techniques have been developed for the identification of drug leads from complex mixtures. In this study, we have developed and optimized a microfluidic online screening technique coupled to nano-liquid chromatography (LC) and in parallel with a mass spectrometer for the identification of thrombin and FXa inhibitors in mixtures. Inhibitors eluting from the nano-LC were split postcolumn in a 1:1 ratio; half was fed into a mass spectrometer (where its mass is detected), and the other half was fed into a microfluidic chip (which acts as a microreactor for the online assays). With our platform, thrombin and FXa inhibitors were detected in the assay in parallel with their mass identification. These methods are suitable for the identification of inhibitors from sample amounts as low as sub-microliter volumes.

  5. Discovery of imidazo[1,5-c]imidazol-3-ones: weakly basic, orally active factor Xa inhibitors.

    PubMed

    Imaeda, Yasuhiro; Kuroita, Takanobu; Sakamoto, Hiroki; Kawamoto, Tetsuji; Tobisu, Mamoru; Konishi, Noriko; Hiroe, Katsuhiko; Kawamura, Masaki; Tanaka, Toshimasa; Kubo, Keiji

    2008-06-26

    The coagulation enzyme factor Xa (FXa) has been recognized as a promising target for the development of new antithrombotic agents. We previously found compound 1 to be an orally bioavailable FXa inhibitor in fasted monkeys; however, 1 showed poor bioavailability in rats and fed monkeys. To work out the pharmacokinetic problems, we focused our synthetic efforts on the chemical conversion of the 4-(imidazo[1,2- a]pyridin-5-yl)piperazine moiety of 1 to imidazolylpiperidine derivatives (fused and nonfused), which resulted in the discovery of the weakly basic imidazo[1,5- c]imidazol-3-one 3q as a potent and selective FXa inhibitor. Compound 3q showed favorable oral bioavailability in rats and monkeys under both fasted and fed conditions and antithrombotic efficacy in a rat model of venous thrombosis after oral administration, without a significant increase in bleeding time (unlike warfarin). On the basis of these promising properties, compound 3q was selected for further evaluation.

  6. Effects of the oral, direct factor Xa inhibitor apixaban on routine coagulation assays and anti-FXa assays.

    PubMed

    Hillarp, A; Gustafsson, K M; Faxälv, L; Strandberg, K; Baghaei, F; Fagerberg Blixter, I; Berndtsson, M; Lindahl, T L

    2014-09-01

    Apixaban is an oral direct factor Xa inhibitor developed for the prophylaxis and treatment of thromboembolic disorders. Laboratory monitoring is not necessary, but the effects on common coagulation reagents and assays constitute clinically valuable information. To investigate the effects of apixaban on commonly used coagulation methods, and to evaluate anti-FXa assays for specific determination of the drug concentration. Apixaban was added to plasma from healthy subjects in the concentration range 0-1000 μg L(-1) , and analyses were performed with different reagents for activated partial thromboplastin time (APTT), prothrombin time (PT), antithrombin, protein C, and protein S. A lupus anticoagulant assay and an APTT assay with varying phospholipid concentrations were used to study the phospholipid dependence. In general, apixaban showed fewer effects in vitro than have been shown for rivaroxaban, another direct FXa inhibitor. The concentration needed to double the APTT varied between 2200 and 4700 μg L(-1) , and the concentration needed to double the PT varied between 700 and 3900 μg L(-1) . The effects on antithrombin, protein C and protein S assays were dependent on the type of reagent. Apixaban did not cause false-positive lupus anticoagulant results. Chromogenic anti-FXa assays showed linear dose-response curves with apixaban. Therapeutic concentrations of apixaban variably affect different assay groups, and even different reagents within an assay group. The effects were much smaller than with rivaroxaban. The use of APTT and/or PT assays to screen the anticoagulant activity of apixaban cannot be recommended. A chromogenic anti-FXa assay can be used for reliable measurements of apixaban concentration. © 2014 International Society on Thrombosis and Haemostasis.

  7. Human plasma kallikrein and tissue kallikrein binding to a substrate based on the reactive site of a factor Xa inhibitor isolated from Bauhinia ungulata seeds.

    PubMed

    Oliva, M L; Andrade, S A; Batista, I F; Sampaio, M U; Juliano, M; Fritz, H; Auerswald, E A; Sampaio, C A

    1999-12-01

    Kunitz type Bauhinia ungulata factor Xa inhibitor (BuXI) was purified from B. ungulata seeds. BuXI inactivates factor Xa and human plasma kallikrein (HuPK) with Ki values of 18.4 and 6.9 nM, respectively. However, Bauhinia variegata trypsin inhibitor (BvTI) which is 70% homologous to BuXI does not inhibit factor Xa and is less efficient on HuPK (Ki = 80 nM). The comparison between BuXI and BvTI reactive site structure indicates differences at Met59, Thr66 and Met67 residues. The hydrolysis rate of quenched fluorescence peptide substrates based on BuXI reactive site sequence, Abz-VMIAALPRTMFIQ-EDDnp (leading peptide), by HuPK and porcine pancreatic kallikrein (PoPK) is low, but hydrolysis is enhanced with Abz-VMIAALPRTMQ-EDDnp, derived from the leading peptide shortened by removing the dipeptide Phe-Ileu from the C-terminal portion, for HuPK (Km = 0.68 microM, k(cat)/Km = 1.3 x 10(6) M(-1) s(-1)), and the shorter substrate Abz-LPRTMQ-EDDnp is better for PoPK (Km = 0.66 microM, k(cat)/Km = 2.2 x 10(3) M(-1) s(-1)). The contribution of substrate methionine residues to HuPK and PoPK hydrolysis differs from that observed with factor Xa. The determined Km and k(cat) values suggest that the substrates interact with kallikreins the same as an enzyme and inhibitor interacts to form complexes.

  8. D-phenylglycinol-derived non-covalent factor Xa inhibitors: effect of non-peptidic S4 linkage elements on affinity and anticoagulant activity.

    PubMed

    Klimkowski, Valentine J; Watson, Brian M; Wiley, Michael R; Liebeschuetz, John; Franciskovich, Jeffry B; Marimuthu, Jothirajah; Bastian, Jolie A; Sall, Daniel J; Smallwood, Jeffrey K; Chirgadze, Nikolay Y; Smith, Gerald F; Foster, Ronald S; Craft, Trelia; Sipes, Philip; Chastain, Marcia; Sheehan, Scott M

    2007-11-01

    Analogs to a series of D-phenylglycinamide-derived factor Xa inhibitors were discovered. It was found that the S4 amide linkage can be replaced with an ether linkage to reduce the peptide character of the molecules and that this substitution leads to an increase in binding affinity that is not predicted based on modeling. Inhibitors which incorporate ether, amino, or alkyl S4 linkage motifs exhibit similar levels of binding affinity and also demonstrate potent in vitro functional activity, however, binding affinity in this series is strongly dependent on the nature of the S1 binding element.

  9. Qualitative and quantitative pharmacophore-similarity assessment of anthranilamide-based factor Xa inhibitors: applications on similar molecules with identical biological endpoints.

    PubMed

    Kumar, Sivakumar Prasanth; Rawal, Rakesh M; Pandya, Himanshu A; Jasrai, Yogesh T

    2016-01-01

    It is a conventional practice to exclude molecules with identical biological endpoints to avoid bias in the resulting hypothesis model. Despite the diverse chemical functionalities, the receptor interactions of such molecules are often unexplored. The present study motivates the selection of these molecules diversified by single atom or functional group compared to internal molecules as external set and helps in the understanding of corresponding effects toward receptor interactions and biological endpoints. Applied on anthranilamide-series of factor Xa analogs, the inhibitory activities were correlated (r(2) = 0.99) and validated (q(2) = 0.68) with distance-based pharmacophore descriptors using support vector machine. The selected external set molecules exhibited better prediction accuracy by securing activities less than one residual threshold. The effect on inhibitory activity was assessed by the examination of pharmacophore-similarity and its interactions with key residues of Human factor Xa enzyme using molecular docking approach. Furthermore, qualitative pharmacophore models were developed on the subset of molecular dataset divided as most actives, moderately actives and least actives, to recognize crucial activity governing pharmacophore features. The outcome of this study will bring new insights about the requirements of pharmacophore features and prioritizes its selection in the design and optimization of potent Xa inhibitors.

  10. The effects of direct factor Xa inhibitor (Rivaroxaban) on the human osteoblastic cell line SaOS2.

    PubMed

    Gigi, Roy; Salai, Moshe; Dolkart, Oleg; Chechik, Ofir; Katzburg, Sarah; Stern, Naftali; Somjen, Dalia

    2012-01-01

    Thromboprophylaxis reduces the risk of surgery-related deep vein thrombosis, but anticoagulants were associated with systemic osteoporosis, a known risk factor for poor fracture healing. Rivaroxaban (XARELTO(®)) is a novel anticoagulant with specific ability to inhibit factor Xa, a serine endopeptidase, which plays a key role in coagulation. This study investigated the direct effects of rivaroxaban on bone biology using an in vitro cell culture model from the human female osteoblastic cell line SaOS2. Cells at subconfluence were treated for 24 hr with different concentrations of rivaroxaban and analyzed for DNA synthesis and creatine kinase- and alkaline phosphatase-specific activities, and were treated 21 days for analyzing mineralization. Rivaroxaban (0.01-50 μg/ml) dose-dependently inhibited up to 60% DNA synthesis of the cells. Creatine kinase-specific activity was also inhibited dose-dependently to a similar extent by the same concentrations. Alkaline phosphatase-specific activity was dose-dependently inhibited but only up to 30%. Cell mineralization was unaffected by 10 μg/ml rivaroxaban. This model demonstrated a significant rivaroxaban-induced reduction in osteoblastic cell growth and energy metabolism, and slight inhibition of the osteoblastic marker, alkaline phosphatase, while osteoblastic mineralization was unaffected. These findings might indicate that rivaroxaban inhibits the first stage of bone formation but does not affect later stages (i.e., bone mineralization).

  11. β-D-glucosyl conjugates of highly potent inhibitors of blood coagulation factor Xa bearing 2-chorothiophene as a P1 motif.

    PubMed

    Lopopolo, Gianfranco; de Candia, Modesto; Panza, Luigi; Romano, Maria Rosaria; Lograno, Marcello Diego; Campagna, Francesco; Altomare, Cosimo

    2012-09-01

    We synthesized a novel O-glucoside of the recently reported potent factor Xa (fXa) inhibitor 1, which bears a 5-chlorothien-2-yl moiety and 1-isopropylpiperidine as fragments that bind the S1 and S4 enzyme pockets, respectively. A β-D-glucosyl unit was conjugated through an ether-linked C3-alkyl spacer to the central phenyl ring of 1. The synthesized β-D-glucose-based compound 16 achieved picomolar inhibitory potency against human fXa (K(i)=60 pM) and high selectivity over thrombin and other serine proteases. In addition to the chlorothienyl S1 binder, a large gain in ΔG resulted from the addition of protonated 1-isopropylpiperidine (ΔΔG=29.7-30.5 kJ mol(-1)), which should bind to the aromatic S4 pocket through efficient cation-π and C-H···π interactions. Instead, the C3-alkyl-linked glucose fragment, which is likely directed toward the solvent outside the enzyme binding site, improves ΔG by an average of 2.9-3.8 kJ mol(-1) . Compound 16 showed sub-micromolar in vitro anticoagulant activity, as assessed by prothrombin time (PT) and activated thromboplastin time (aPTT) clotting assays in pooled human plasma (PT(2) and aPTT(2) equal to 0.135 and 0.389 μM, respectively). Although compound 16 was 1.4-fold less active than parent compound 1 in the ex vivo anticoagulant assay in mice, it showed a significant (1.6-fold) prolongation of PT relative to controls (P<0.05) 60 min after oral dosing (75 mg kg(-1)). Copyright © 2012 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  12. CrataBL, a lectin and Factor Xa inhibitor, plays a role in blood coagulation and impairs thrombus formation.

    PubMed

    Salu, Bruno R; Ferreira, Rodrigo S; Brito, Marlon V; Ottaiano, Tatiana F; Cruz, José Walber M C; Silva, Mariana Cristina C; Correia, Maria Tereza S; Paiva, Patrícia M G; Maffei, Francisco Humberto A; Oliva, Maria Luiza Vilela

    2014-09-01

    Arterial thrombosis is an important complication of diabetes and cancer, being an important target for therapeutic intervention. Crataeva tapia bark lectin (CrataBL) has been previously shown to have hypoglycemiant effect and also to induce cancer cell apoptosis. It also showed inhibitory activity against Factor Xa (Kiapp=8.6 μm). In the present study, we evaluated the anti-thrombotic properties of CrataBL in arterial thrombosis model. CrataBL prolongs the activated partial thromboplastin time on human and mouse plasma, and it impairs the heparin-induced potentiation of antithrombin III and heparin-induced platelet activation in the presence of low-dose ADP. It is likely that the dense track of positive charge on CrataBL surface competes with the heparin ability to bind to antithrombin III and to stimulate platelets. In the photochemically induced thrombosis model in mice, in the groups treated with 1.25, 5.0, or 10 mg/kg CrataBL, prior to the thrombus induction, the time of total artery occlusion was prolonged by 33.38%, 65%, and 66.11%, respectively, relative to the time of the control group. In contrast to heparin, the bleeding time in CrataBL-treated mice was no longer than in the control. In conclusion, CrataBL was effective in blocking coagulation and arterial thrombus formation, without increasing bleeding time.

  13. Conserved Amblyomma americanum tick Serpin19, an inhibitor of blood clotting factors Xa and XIa, trypsin and plasmin, has anti-haemostatic functions.

    PubMed

    Kim, Tae Kwon; Tirloni, Lucas; Radulovic, Zeljko; Lewis, Lauren; Bakshi, Mariam; Hill, Creston; da Silva Vaz, Itabajara; Logullo, Carlos; Termignoni, Carlos; Mulenga, Albert

    2015-08-01

    Tick saliva serine protease inhibitors (serpins) facilitate tick blood meal feeding through inhibition of protease mediators of host defense pathways. We previously identified a highly conserved Amblyomma americanum serpin 19 that is characterised by its reactive center loop being 100% conserved in ixodid ticks. In this study, biochemical characterisation reveals that the ubiquitously transcribed A. americanum serpin 19 is an anti-coagulant protein, inhibiting the activity of five of the eight serine protease blood clotting factors. Pichia pastoris-expressed recombinant (r) A. americanum serpin 19 inhibits the enzyme activity of trypsin, plasmin and blood clotting factors (f) Xa and XIa, with stoichiometry of inhibition estimated at 5.1, 9.4, 23.8 and 28, respectively. Similar to typical inhibitory serpins, recombinant A. americanum serpin 19 forms irreversible complexes with trypsin, fXa and fXIa. At a higher molar excess of recombinant A. americanum serpin 19, fXIIa is inhibited by 82.5%, and thrombin (fIIa), fIXa, chymotrypsin and tryptase are inhibited moderately by 14-29%. In anti-hemostatic functional assays, recombinant A. americanum serpin 19 inhibits thrombin but not ADP and cathepsin G activated platelet aggregation, delays clotting in recalcification and thrombin time assays by up to 250s, and up to 40s in the activated partial thromboplastin time assay. Given A. americanum serpin 19 high cross-tick species conservation, and specific reactivity of recombinant A. americanum serpin 19 with antibodies to A. americanum tick saliva proteins, we conclude that recombinant A. americanum serpin 19 is a potential candidate for development of a universal tick vaccine.

  14. The discovery of (2R,4R)-N-(4-chlorophenyl)-N- (2-fluoro-4-(2-oxopyridin-1(2H)-yl)phenyl)-4-methoxypyrrolidine-1,2-dicarboxamide (PD 0348292), an orally efficacious factor Xa inhibitor.

    PubMed

    Kohrt, Jeffrey T; Bigge, Christopher F; Bryant, John W; Casimiro-Garcia, Agustin; Chi, Liguo; Cody, Wayne L; Dahring, Tawny; Dudley, Danette A; Filipski, Kevin J; Haarer, Staci; Heemstra, Ron; Janiczek, Nancy; Narasimhan, Lakshmi; McClanahan, Thomas; Peterson, J Thomas; Sahasrabudhe, Vaisheli; Schaum, Robert; Van Huis, Chad A; Welch, Kathleen M; Zhang, Erli; Leadley, Robert J; Edmunds, Jeremy J

    2007-08-01

    Herein, we report the discovery of novel, proline-based factor Xa inhibitors containing a neutral P1 chlorophenyl pharmacophore. Through the additional incorporation of 1-(4-amino-3-fluoro-phenyl)-1H-pyridin-2-one 22, as a P4 pharmacophore, we discovered compound 7 (PD 0348292). This compound is a selective, orally bioavailable, efficacious FXa inhibitor that is currently in phase II clinical trials for the treatment and prevention of thrombotic disorders.

  15. Factor Xa inhibitors: S1 binding interactions of a series of N-{(3S)-1-[(1S)-1-methyl-2-morpholin-4-yl-2-oxoethyl]-2-oxopyrrolidin-3-yl}sulfonamides.

    PubMed

    Chan, Chuen; Borthwick, Alan D; Brown, David; Burns-Kurtis, Cynthia L; Campbell, Matthew; Chaudry, Laiq; Chung, Chun-wa; Convery, Máire A; Hamblin, J Nicole; Johnstone, Lisa; Kelly, Henry A; Kleanthous, Savvas; Patikis, Angela; Patel, Champa; Pateman, Anthony J; Senger, Stefan; Shah, Gita P; Toomey, John R; Watson, Nigel S; Weston, Helen E; Whitworth, Caroline; Young, Robert J; Zhou, Ping

    2007-04-05

    Factor Xa inhibitory activities for a series of N-{(3S)-1-[(1S)-1-methyl-2-morpholin-4-yl-2-oxoethyl]-2-oxopyrrolidin-3-yl}sulfonamides with different P1 groups are described. These data provide insight into binding interactions within the S1 primary specificity pocket; rationales are presented for the derived SAR on the basis of electronic interactions through crystal structures of fXa-ligand complexes and molecular modeling studies. A good correlation between in vitro anticoagulant activities with lipophilicity and the extent of human serum albumin binding is observed within this series of potent fXa inhibitors. Pharmacokinetic profiles in rat and dog, together with selectivity over other trypsin-like serine proteases, identified 1f as a candidate for further evaluation.

  16. OC-03 - Modelisation of the procoagulant properties of cancer cells and their capacity to alter the antithrombotic efficiency of LMWHs and specific inhibitors of factor Xa.

    PubMed

    Gerotziafas, G T; Rousseau, A; van Dreden, P; Mbemba, E; Gkalea, V; Khaterchi, A; Larsen, A; Elalamy, I

    2016-04-01

    The risk of venous thromboembolism varies according to the histological type of cancer. The failure of antithrombotic treatment is more frequent in cancer patients as compared to non-cancer ones. We aimed to elucidate the mechanism of activation of blood coagulation induced by cancer, the impact of chemo-resistance phenotype on the capacity of cancer cells to trigger thrombin generation and the alterations of the efficiency of LMWHs and the specific inhibitors of factor Xa (fondaparinux and apixaban) in the presence of cancer cells. Thrombin generation of human plasma was assessed in the presence of various cancer cell lines. The model of cancer-induced hypercoagulability was coupled to the research for the expression of procoagulant molecules by cancer cells. The pancreatic adenocarcinoma cells BXPC3 and the breast adenocarcinoma cells MCF7 were initially tested. The BXPC3 cells induce significantly higher thrombin generation as compared to the MCF7 cells. In the same line Marchetti et al. showed that malignant hematologic cells (NB4, HEL, and K562) and H69 small cell lung cells express different procoagulant potential on triggering thrombin generation of human plasma. The comparison of the procoagulant activity has been extended to cancer cell lines from various cancers (i.e. colon, ovarian and prostatic cancer) as well as to different cell lines of the same type of cancer. The differences of the cancer cell lines to trigger thrombin generation are mainly due to the expression of TF. The acquisition of chemoresistant phenotype by cancer cells is correlated with increased TF expression and enhancement of theit procoagulant activity. The ability of cancer cells to activate FXII is an alternative pathway of significant importance for some cancer cell lines (i.e. MCF7). Clinically relevant concentrations of LMWH and specific direct and indirect inhibitors of FXa (apixaban and fondaparinux) inhibit thrombin generation induced by cancer cells. The synergy between the

  17. Lufaxin, a Novel Factor Xa Inhibitor from the Salivary Gland of the sand fly Lutzomyia longipalpis, Blocks PAR2 Activation and Inhibits Inflammation and Thrombosis in Vivo

    PubMed Central

    Collin, Nicolas; Assumpção, Teresa C. F.; Mizurini, Daniella M.; Gilmore, Dana; Dutra-Oliveira, Angélica; Kotsyfakis, Michalis; Sá-Nunes, Anderson; Teixeira, Clarissa; Ribeiro, José M. C.; Monteiro, Robson Q.; Valenzuela, Jesus G.; Francischetti, Ivo M. B.

    2012-01-01

    Objective Blood-sucking arthropods salivary glands (SGs) contain a remarkable diversity of antihemostatics. The aim of this study was to identify the unique salivary anticoagulant of the sand fly Lutzomyia longipalpis, which remained elusive for decades. Methods and Results Several L. longipalpis salivary proteins were expressed in HEK293 cells and screened for inhibition of blood coagulation. A novel 32.4-kDa molecule, named Lufaxin, was identified as a slow, tight, non-competitive, and reversible inhibitor of Factor Xa (FXa). Notably, Lufaxin primary sequence does not share similarity to any physiological or salivary inhibitors of coagulation reported to date. Lufaxin is specific for FXa and does not interact with FX, DEGR- FXa, or 15 other enzymes. In addition, Lufaxin blocks prothrombinase and increases both PT and aPTT. Surface plasmon resonance experiments revealed that FXa binds Lufaxin with a KD ~3 nM, and isothermal titration calorimetry determined a stoichiometry of 1:1. Lufaxin also prevents PAR2 activation by FXa in the MDA-MB-231 cell line and abrogates edema formation triggered by injection of FXa in the paw of mice. Moreover, Lufaxin prevents FeCl3-induced carotid artery thrombus formation and prolongs aPTT ex vivo, implying that it works as an anticoagulant in vivo. Finally, SG of sandflies was found to inhibit FXa and to interact with the enzyme. Conclusion Lufaxin belongs to a novel family of slow-tight FXa inhibitors, which display antithrombotic and antiinflamatory activities. It is a useful tool to understand FXa structural features and its role in pro-hemostatic and pro-inflammatory events. PMID:22796577

  18. Computational investigation of potential dosing schedules for a switch of medication from warfarin to rivaroxaban—an oral, direct Factor Xa inhibitor

    PubMed Central

    Burghaus, Rolf; Coboeken, Katrin; Gaub, Thomas; Niederalt, Christoph; Sensse, Anke; Siegmund, Hans-Ulrich; Weiss, Wolfgang; Mueck, Wolfgang; Tanigawa, Takahiko; Lippert, Jörg

    2014-01-01

    The long-lasting anticoagulant effect of vitamin K antagonists can be problematic in cases of adverse drug reactions or when patients are switched to another anticoagulant therapy. The objective of this study was to examine in silico the anticoagulant effect of rivaroxaban, an oral, direct Factor Xa inhibitor, combined with the residual effect of discontinued warfarin. Our simulations were based on the recommended anticoagulant dosing regimen for stroke prevention in patients with atrial fibrillation. The effects of the combination of discontinued warfarin plus rivaroxaban were simulated using an extended version of a previously validated blood coagulation computer model. A strong synergistic effect of the two distinct mechanisms of action was observed in the first 2–3 days after warfarin discontinuation; thereafter, the effect was close to additive. Nomograms for the introduction of rivaroxaban therapy after warfarin discontinuation were derived for Caucasian and Japanese patients using safety and efficacy criteria described previously, together with the coagulation model. The findings of our study provide a mechanistic pharmacologic rationale for dosing schedules during the therapy switch from warfarin to rivaroxaban and support the switching strategies as outlined in the Summary of Product Characteristics and Prescribing Information for rivaroxaban. PMID:25426077

  19. A computational modeling and molecular dynamics study of the Michaelis complex of human protein Z-dependent protease inhibitor (ZPI) and factor Xa (FXa)

    PubMed Central

    Chandrasekaran, Vasudevan; Lee, Chang Jun; Lin, Ping; Duke, Robert E.

    2009-01-01

    Protein Z-dependent protease inhibitor (ZPI) and antithrombin III (AT3) are members of the serpin superfamily of protease inhibitors that inhibit factor Xa (FXa) and other proteases in the coagulation pathway. While experimental structural information is available for the interaction of AT3 with FXa, at present there is no structural data regarding the interaction of ZPI with FXa, and the precise role of this interaction in the blood coagulation pathway is poorly understood. In an effort to gain a structural understanding of this system, we have built a solvent equilibrated three-dimensional structural model of the Michaelis complex of human ZPI/FXa using homology modeling, protein–protein docking and molecular dynamics simulation methods. Preliminary analysis of interactions at the complex interface from our simulations suggests that the interactions of the reactive center loop (RCL) and the exosite surface of ZPI with FXa are similar to those observed from X-ray crystal structure-based simulations of AT3/FXa. However, detailed comparison of our modeled structure of ZPI/FXa with that of AT3/FXa points to differences in interaction specificity at the reactive center and in the stability of the inhibitory complex, due to the presence of a tyrosine residue at the P1 position in ZPI, instead of the P1 arginine residue in AT3. The modeled structure also shows specific structural differences between AT3 and ZPI in the heparin-binding and flexible N-terminal tail regions. Our structural model of ZPI/FXa is also compatible with available experimental information regarding the importance for the inhibitory action of certain basic residues in FXa. PMID:19172319

  20. The effect of the direct factor Xa inhibitors apixaban and rivaroxaban on haemostasis tests: a comprehensive assessment using in vitro and ex vivo samples.

    PubMed

    Bonar, Roslyn; Favaloro, Emmanuel J; Mohammed, Soma; Ahuja, Monica; Pasalic, Leonardo; Sioufi, John; Marsden, Katherine

    2016-01-01

    The direct oral anticoagulants (DOACs), now including dabigatran, apixaban and rivaroxaban, have given clinicians alternative options to low molecular weight heparins (LMWHs) and vitamin K antagonist therapy, including warfarin, for the treatment of atrial fibrillation and treatment and prevention of venous thromboembolic (VTE) disease. DOACs have been successfully marketed as not requiring monitoring; however, there will be situations where clinicians will request laboratory testing, including emergency department admissions for haemorrhage or thrombosis, or emergency surgical interventions. We report the results of several Royal College of Pathologists of Australasia Quality Assurance Programs (RCPAQAP) surveys using apixaban and rivaroxaban spiked samples to either assess the suitability of certain potential screening or drug-quantifying assays, for assessment of drug presence or absence or measurement of levels, as well as assessing potential interference in a wide variety of haemostasis assays. We also include additional evaluations using ex vivo samples from patients given apixaban and rivaroxaban for various therapeutic reasons. The prothrombin time (PT) and activated partial thromboplastin time (APTT) show better sensitivity with rivaroxaban than apixaban. Anti-Xa assays show good concordance and reproducibility with expected drug levels; however, availability of these assays may be limited to larger institutions. Interference of apixaban and rivaroxaban on haemostasis testing extends beyond routine coagulation assays to encompass a plethora of specialised assays, including factor assays, lupus inhibitor, and FVIII inhibitor estimation. In conclusion, this report highlights the influence of these drugs on most tests performed in haemostasis laboratories, and the potential for some tests to predict the presence, absence or level of these drugs in plasma.

  1. Synergistic effect of a factor Xa inhibitor, TAK-442, and antiplatelet agents on whole blood coagulation and arterial thrombosis in rats.

    PubMed

    Konishi, Noriko; Hiroe, Katsuhiko; Kawamura, Masaki

    2010-08-01

    Activated platelets facilitate blood coagulation by providing factor V and a procoagulant surface for prothrombinase. Here, we investigated the potential synergy of a potent factor Xa/prothrombinase inhibitor, TAK-442, plus aspirin or clopidogrel in preventing arterial thrombosis and whole blood coagulation. Thrombus formation was initiated by FeCl(3)-induced rat carotid injury. Bleeding time was evaluated with the rat tail transection model. Whole blood coagulation was assessed by thromboelastographic examination (TEG) for which blood obtained from control, aspirin-, or clopidogrel-treated rats was transferred to a TEG analyzer containing, collagen or adenosine diphosphate (ADP), and TAK-442 or vehicle. TAK-442 (3mg/kg, po), aspirin (100mg/kg, po) or clopidogrel (3mg/kg, po) alone had no significant effect on thrombus formation, whereas the combination of TAK-442 with aspirin and clopidogrel remarkably prolonged the time to thrombus formation without additional significant prolongation of bleeding time. TEG demonstrated that the onset of collagen-induced blood coagulation were slightly longer in aspirin-treated rats than control; however, when the blood from aspirin-treated rats was subsequently treated in vitro with 100 nM TAK-442, the onset of clotting was significantly prolonged. In contrast, only marginal prolongation was observed with TAK-442 treatment of blood from control animals. The onset time of ADP-induced blood coagulation was slightly longer in clopidogrel-treated rats compared with control, and it was further extended by TAK-442 treatment. These results demonstrate that blood coagulation can be markedly delayed by the addition of TAK-442 to antiplatelets treatment which could contribute to synergistic antithrombotic efficacy in these settings. (c) 2010 Elsevier Ltd. All rights reserved.

  2. Safety and efficacy of edoxaban, an oral factor Xa inhibitor, versus enoxaparin for thromboprophylaxis after total knee arthroplasty: the STARS E-3 trial.

    PubMed

    Fuji, Takeshi; Wang, Ching-Jen; Fujita, Satoru; Kawai, Yohko; Nakamura, Mashio; Kimura, Tetsuya; Ibusuki, Kei; Ushida, Hitoshi; Abe, Kenji; Tachibana, Shintaro

    2014-12-01

    This phase 3 trial compared the safety and efficacy of edoxaban, an oral direct factor Xa inhibitor, with enoxaparin sodium (enoxaparin) for thromboprophylaxis after total knee arthroplasty (TKA) in patients in Japan and Taiwan. In this randomized, double-blind, double-dummy study, patients received oral edoxaban 30 mg once daily beginning 6 to 24 hours postsurgery or enoxaparin 2000 IU (equivalent to 20 mg) subcutaneously twice daily beginning 24 to 36 hours postsurgery for 11 to 14 days. The primary efficacy endpoint was the composite of symptomatic pulmonary embolism and symptomatic and asymptomatic deep vein thrombosis. Safety endpoints included the incidence of major bleeding, clinically relevant non-major (CRNM) bleeding, major bleeding or CRNM bleeding, all bleeding events, adverse events, and adverse drug reactions. Of 716 patients enrolled, 360 and 356 were randomized to receive edoxaban or enoxaparin, respectively. The primary efficacy outcome occurred in 22/299 (7.4%) and 41/295 (13.9%) patients in the edoxaban and enoxaparin groups, respectively (relative risk reduction=46.8%), indicating non-inferiority (P <0.001) and superiority (P=0.010) of edoxaban versus enoxaparin. In the edoxaban and enoxaparin groups, major bleeding occurred in 4/354 (1.1%) versus 1/349 (0.3%) patients (P=0.373); major or CRNM bleeding occurred in 22/354 (6.2%) versus 13/349 (3.7%) patients (P=0.129), respectively. Edoxaban 30 mg once daily was more effective for thromboprophylaxis than subcutaneous enoxaparin 2000 IU twice daily following TKA and demonstrated a similar incidence of bleeding events. Copyright © 2014 Elsevier Ltd. All rights reserved.

  3. Rivaroxaban, a direct inhibitor of the coagulation factor Xa interferes with hormonal-induced physiological modulations in human female osteoblastic cell line SaSO2.

    PubMed

    Somjen, Dalia; Katzburg, Sara; Gigi, Roi; Dolkart, Oleg; Sharon, Orli; Salai, Moshe; Stern, Naftali

    2013-05-01

    The use of anticoagulants has been associated with systemic osteoporosis and increased risk for poor fracture healing but is inevitable following major orthopedic surgery of lower limbs. Rivaroxaban A (R) is an anticoagulant recently introduced in the clinical setting, which is a specific factor Xa inhibitor. We reported previously that R significantly inhibited cell growth, energy metabolism and alkaline phosphatase activity in human osteoblastic cell line SaOS2, with no effect on mineralization, indicating transient inhibition of bone formation. We now investigated the effects of R on SaOS2 response to osteoblast-modulating hormones. At sub-confluence cells were treated with: estradiol-17β (E2), the phytoestrogens daidzein (D) and biochainin A (BA), the carboxy-pytoestrogenic derivative carboxy-D (cD), the estrogen receptor α (ERα) agonist PPT, the estrogen receptor β (ERβ) agonist DPN, parathyroid hormone (PTH) and several vitamin D metabolites and analogs with/without R for 24h. All hormones tested stimulated significantly DNA synthesis (DNA), creatine kinase (CK) and alkaline phosphatase (ALP) specific activities, but all these stimulations were totally inhibited when given together with R. R had no effect on mRNA expression of ERα, ERβ and 25 Hydroxy-vitamin D3-1α hydroxylase (1OHase), but inhibited hormonal modulations of mRNA expressions. In conclusion R inhibited significantly hormonal stimulation of different parameters indicating inhibition of not only the early stages of bone formation, but also the stimulatory effects of bone modulating hormones with a yet unclear mechanism. The relevance of these findings to human bone physiology is yet to be investigated.

  4. Home treatment of patients with low-risk pulmonary embolism with the oral factor Xa inhibitor rivaroxaban. Rationale and design of the HoT-PE Trial.

    PubMed

    Barco, Stefano; Lankeit, Mareike; Binder, Harald; Schellong, Sebastian; Christ, Michael; Beyer-Westendorf, Jan; Duerschmied, Daniel; Bauersachs, Rupert; Empen, Klaus; Held, Matthias; Schwaiblmair, Martin; Fonseca, Cândida; Jiménez, David; Becattini, Cecilia; Quitzau, Kurt; Konstantinides, Stavros

    2016-07-04

    Pulmonary embolism (PE) is a potentially life-threatening acute cardiovascular syndrome. However, more than 95 % of patients are haemodynamically stable at presentation, and among them are patients at truly low risk who may qualify for immediate or early discharge. The Home Treatment of Pulmonary Embolism (HoT-PE) study is a prospective international multicentre single-arm phase 4 management (cohort) trial aiming to determine whether home treatment of acute low-risk PE with the oral factor Xa inhibitor rivaroxaban is feasible, effective, and safe. Patients with confirmed PE, who have no right ventricular dysfunction or free floating thrombi in the right atrium or ventricle, are eligible if they meet none of the exclusion criteria indicating haemodynamic instability, serious comorbidity or any condition mandating hospitalisation, or a familial/social environment unable to support home treatment. The first dose of rivaroxaban is given in hospital, and patients are discharged within 48 hours of presentation. Rivaroxaban is taken for at least three months. The primary outcome is symptomatic recurrent venous thromboembolism or PE-related death within three months of enrolment. Secondary outcomes include quality of life and patient satisfaction, and health care resource utilisation compared to existing data on standard-duration hospital treatment. HoT-PE is planned to analyse 1,050 enrolled patients, providing 80 % power to reject the null hypothesis that the recurrence rate of venous thromboembolism is >3 % with α≤0.05. If the hypothesis of HoT-PE is confirmed, early discharge and out-of-hospital treatment may become an attractive, potentially cost-saving option for a significant proportion of patients with acute PE.

  5. At-line nanofractionation with parallel mass spectrometry and bioactivity assessment for the rapid screening of thrombin and factor Xa inhibitors in snake venoms.

    PubMed

    Mladic, Marija; Zietek, Barbara M; Iyer, Janaki Krishnamoorthy; Hermarij, Philip; Niessen, Wilfried M A; Somsen, Govert W; Kini, R Manjunatha; Kool, Jeroen

    2016-02-01

    Snake venoms comprise complex mixtures of peptides and proteins causing modulation of diverse physiological functions upon envenomation of the prey organism. The components of snake venoms are studied as research tools and as potential drug candidates. However, the bioactivity determination with subsequent identification and purification of the bioactive compounds is a demanding and often laborious effort involving different analytical and pharmacological techniques. This study describes the development and optimization of an integrated analytical approach for activity profiling and identification of venom constituents targeting the cardiovascular system, thrombin and factor Xa enzymes in particular. The approach developed encompasses reversed-phase liquid chromatography (RPLC) analysis of a crude snake venom with parallel mass spectrometry (MS) and bioactivity analysis. The analytical and pharmacological part in this approach are linked using at-line nanofractionation. This implies that the bioactivity is assessed after high-resolution nanofractionation (6 s/well) onto high-density 384-well microtiter plates and subsequent freeze drying of the plates. The nanofractionation and bioassay conditions were optimized for maintaining LC resolution and achieving good bioassay sensitivity. The developed integrated analytical approach was successfully applied for the fast screening of snake venoms for compounds affecting thrombin and factor Xa activity. Parallel accurate MS measurements provided correlation of observed bioactivity to peptide/protein masses. This resulted in identification of a few interesting peptides with activity towards the drug target factor Xa from a screening campaign involving venoms of 39 snake species. Besides this, many positive protease activity peaks were observed in most venoms analysed. These protease fingerprint chromatograms were found to be similar for evolutionary closely related species and as such might serve as generic snake protease

  6. Factor Xa stimulates fibroblast procollagen production, proliferation, and calcium signaling via PAR{sub 1} activation

    SciTech Connect

    Blanc-Brude, Olivier P. . E-mail: olivier.blanc-brude@larib.inserm.fr; Archer, Fabienne; Leoni, Patricia; Derian, Claudia; Bolsover, Steven; Laurent, Geoffrey J.; Chambers, Rachel C.

    2005-03-10

    Fibroblast proliferation and procollagen production are central features of tissue repair and fibrosis. In addition to its role in blood clotting, the coagulation cascade proteinase thrombin can contribute to tissue repair by stimulating fibroblasts via proteolytic activation of proteinase-activated receptor-1 (PAR{sub 1}). During hemostasis, the coagulation cascade proteinase factor X is converted into factor Xa. We have previously shown that factor Xa upregulates fibroblast proliferation via production of autocrine PDGF. In this study, we further examined the effects of factor Xa on fibroblast function and aimed to identify its signaling receptor. We showed that factor Xa stimulates procollagen promoter activity and protein production by human and mouse fibroblasts. This effect was independent of PDGF and thrombin production, but dependent on factor Xa proteolytic activity. We also showed that PAR{sub 1}-deficient mouse fibroblasts did not upregulate procollagen production, mobilize cytosolic calcium, or proliferate in response to factor Xa. Desensitization techniques and PAR{sub 1}-specific agonists and inhibitors were used to demonstrate that PAR{sub 1} mediates factor Xa signaling in human fibroblasts. This is the first report that factor Xa stimulates extracellular matrix production. In contrast with endothelial cells and vascular smooth muscle cells, fibroblasts appear to be the only cell type in which the effects of factor Xa are mediated mainly via PAR{sub 1} and not PAR{sub 2}. These findings are critical for our understanding of tissue repair and fibrotic mechanisms, and for the design of novel approaches to inhibit the profibrotic effects of the coagulation cascade without compromising blood hemostasis.

  7. Discovery of 1-[3-(aminomethyl)phenyl]-N-3-fluoro-2'-(methylsulfonyl)-[1,1'-biphenyl]-4-yl]-3-(trifluoromethyl)-1H-pyrazole-5-carboxamide (DPC423), a highly potent, selective, and orally bioavailable inhibitor of blood coagulation factor Xa.

    PubMed

    Pinto, D J; Orwat, M J; Wang, S; Fevig, J M; Quan, M L; Amparo, E; Cacciola, J; Rossi, K A; Alexander, R S; Smallwood, A M; Luettgen, J M; Liang, L; Aungst, B J; Wright, M R; Knabb, R M; Wong, P C; Wexler, R R; Lam, P Y

    2001-02-15

    Factor Xa (fXa) plays a critical role in the coagulation cascade, serving as the point of convergence of the intrinsic and extrinsic pathways. Together with nonenzymatic cofactor Va and Ca2+ on the phospholipid surface of platelets or endothelial cells, factor Xa forms the prothrombinase complex, which is responsible for the proteolysis of prothrombin to catalytically active thrombin. Thrombin, in turn, catalyzes the cleavage of fibrinogen to fibrin, thus initiating a process that ultimately leads to clot formation. Recently, we reported on a series of isoxazoline and isoxazole monobasic noncovalent inhibitors of factor Xa which show good potency in animal models of thrombosis. In this paper, we wish to report on the optimization of the heterocyclic core, which ultimately led to the discovery of a novel pyrazole SN429 (2b; fXa K(i) = 13 pM). We also report on our efforts to improve the oral bioavailability and pharmacokinetic profile of this series while maintaining subnanomolar potency and in vitro selectivity. This was achieved by replacing the highly basic benzamidine P1 with a less basic benzylamine moiety. Further optimization of the pyrazole core substitution and the biphenyl P4 culminated in the discovery of DPC423 (17h), a highly potent, selective, and orally active factor Xa inhibitor which was chosen for clinical development.

  8. Comparative evaluation of direct thrombin and factor Xa inhibitors with antiplatelet agents under flow and static conditions: an in vitro flow chamber model.

    PubMed

    Hosokawa, Kazuya; Ohnishi, Tomoko; Sameshima, Hisayo; Miura, Naoki; Koide, Takehiko; Maruyama, Ikuro; Tanaka, Kenichi A

    2014-01-01

    Dabigatran and rivaroxaban are novel oral anticoagulants that specifically inhibit thrombin and factor Xa, respectively. The aim of this study is to elucidate antithrombotic properties of these anticoagulant agents under arterial and venous shear conditions. Whole blood samples treated with dabigatran or rivaroxaban at 250, 500, and 1000 nM, with/without aspirin and AR-C66096, a P2Y12 antagonist, were perfused over a microchip coated with collagen and tissue thromboplastin at shear rates of 240 and 600 s(-1). Fibrin-rich platelet thrombus formation was quantified by monitoring flow pressure changes. Dabigatran at higher concentrations (500 and 1000 nM) potently inhibited thrombus formation at both shear rates, whereas 1000 nM of rivaroxaban delayed, but did not completely inhibit, thrombus formation. Dual antiplatelet agents weakly suppressed thrombus formation at both shear rates, but intensified the anticoagulant effects of dabigatran and rivaroxaban. The anticoagulant effects of dabigatran and rivaroxaban were also evaluated under static conditions using thrombin generation (TG) assay. In platelet-poor plasma, dabigatran at 250 and 500 nM efficiently prolonged the lag time (LT) and moderately reduce peak height (PH) of TG, whereas rivaroxaban at 250 nM efficiently prolonged LT and reduced PH of TG. In platelet-rich plasma, however, both anticoagulants efficiently delayed LT and reduced PH of TG. Our results suggest that dabigatran and rivaroxaban may exert distinct antithrombotic effects under flow conditions, particularly in combination with dual antiplatelet therapy.

  9. Comparative Evaluation of Direct Thrombin and Factor Xa Inhibitors with Antiplatelet Agents under Flow and Static Conditions: An In Vitro Flow Chamber Model

    PubMed Central

    Hosokawa, Kazuya; Ohnishi, Tomoko; Sameshima, Hisayo; Miura, Naoki; Koide, Takehiko; Maruyama, Ikuro; Tanaka, Kenichi A.

    2014-01-01

    Dabigatran and rivaroxaban are novel oral anticoagulants that specifically inhibit thrombin and factor Xa, respectively. The aim of this study is to elucidate antithrombotic properties of these anticoagulant agents under arterial and venous shear conditions. Whole blood samples treated with dabigatran or rivaroxaban at 250, 500, and 1000 nM, with/without aspirin and AR-C66096, a P2Y12 antagonist, were perfused over a microchip coated with collagen and tissue thromboplastin at shear rates of 240 and 600 s−1. Fibrin-rich platelet thrombus formation was quantified by monitoring flow pressure changes. Dabigatran at higher concentrations (500 and 1000 nM) potently inhibited thrombus formation at both shear rates, whereas 1000 nM of rivaroxaban delayed, but did not completely inhibit, thrombus formation. Dual antiplatelet agents weakly suppressed thrombus formation at both shear rates, but intensified the anticoagulant effects of dabigatran and rivaroxaban. The anticoagulant effects of dabigatran and rivaroxaban were also evaluated under static conditions using thrombin generation (TG) assay. In platelet-poor plasma, dabigatran at 250 and 500 nM efficiently prolonged the lag time (LT) and moderately reduce peak height (PH) of TG, whereas rivaroxaban at 250 nM efficiently prolonged LT and reduced PH of TG. In platelet-rich plasma, however, both anticoagulants efficiently delayed LT and reduced PH of TG. Our results suggest that dabigatran and rivaroxaban may exert distinct antithrombotic effects under flow conditions, particularly in combination with dual antiplatelet therapy. PMID:24497951

  10. The antithrombotic activity of EP224283, a neutralizable dual factor Xa inhibitor/glycoprotein IIbIIIa antagonist, exceeds that of the coadministered parent compounds.

    PubMed

    Hechler, Béatrice; Freund, Monique; Alame, Ghina; Leguay, Cécile; Gaertner, Sébastien; Cazenave, Jean-Pierre; Petitou, Maurice; Gachet, Christian

    2011-08-01

    EP224283 combines in a single molecule idraparinux and tirofiban, which allows obtaining a predictable and sustained antiplatelet effect through the transfer of the pharmacokinetics properties of idraparinux to the anti-αIIbβ3 antagonist. The activity can be instantaneously neutralized by injection of avidin, a specific antidote. We have tested the effects of this new profile anticoagulant in various thrombosis models. The antithrombotic effect of EP224283 was compared with those of the parent compounds used alone or in association at doses achieving low to moderate inhibition of platelet aggregation ex vivo. In a model of systemic thromboembolism independent of thrombin generation, tirofiban and EP224283 had similar effects at equimolar doses. On the other hand, EP224283 was more potent than tirofiban or idraparinux under thrombin-dependent conditions. In a ferric chloride-induced thrombosis model, EP224283 was more potent than either parent compound or their combination. Similar results were obtained after atherosclerotic plaque rupture in ApoE(-/-) mice. Thus, the dual action of EP224283 exceeds that of the parent compounds used in combination. A possible explanation is that EP224283 could concentrate antithrombin inside the thrombus by binding to αIIbβ3 through the tirofiban moiety, as shown by immunolabeling of the occluded vessel. No prolongation of the bleeding time was observed at doses achieving strong antithrombotic effects, suggesting that low to moderate αIIbβ3 inhibition combined with factor Xa inhibition minimizes the bleeding risk. The favorable antithrombotic profile of EP224283 together with its possible neutralization by avidin makes it an interesting drug candidate for the treatment and prevention of acute ischemic events.

  11. Thrombin generation induced by tissue factor plus ADP in human platelet rich plasma: A potential new measurement to assess the effect of the concomitant use of an oral factor Xa inhibitor edoxaban and P2Y12 receptor antagonists.

    PubMed

    Honda, Yuko; Morishima, Yoshiyuki

    2015-05-01

    Patients with atrial fibrillation undergoing percutaneous coronary intervention may require combination therapy with anticoagulants and antiplatelet agents. The objectives of this study were to establish an assay which can evaluate the effects of both anticoagulants and P2Y12 receptor antagonists and determine the effects of edoxaban, a direct factor Xa inhibitor, and P2Y12 receptor antagonists (clopidogrel and ticagrelor) alone and when combined. Human platelet-rich plasma (PRP) from healthy subjects was stimulated with adenosine diphosphate (ADP) plus tissue factor. Thrombin generation was measured by means of calibrated automated thrombography. Combination of 10μM ADP and low concentration (0.25 pM) tissue factor induced reproducible thrombin generation in human PRP. Edoxaban (40 and 80ng/mL), active metabolite of clopidogrel (AM-clopidogrel, 10 and 20μg/mL), and ticagrelor (3μg/mL) alone inhibited ADP plus tissue factor-induced thrombin generation. Edoxaban suppressed all 5 parameters (lag time, peak, time to peak, endogenous thrombin potential, and maximum rate), whereas AM-clopidogrel and ticagrelor inhibited 4 and 3 parameters, respectively. Concomitant treatment with edoxaban and AM-clopidogrel or ticagrelor produced an additive inhibition of thrombin generation compared to the single treatments. The thrombin generation assay induced by ADP plus tissue factor can detect the activities of both edoxaban and P2Y12 receptor antagonists. Combination of edoxaban and a P2Y12 receptor antagonist shows additive inhibition. These results suggest that ADP plus tissue factor-induced thrombin generation may be a useful measurement to assess the combination effects of anticoagulants and P2Y12 receptor antagonists in a single assay. Copyright © 2015 Elsevier Ltd. All rights reserved.

  12. 1-[3-Aminobenzisoxazol-5'-yl]-3-trifluoromethyl-6-[2'-(3-(R)-hydroxy-N-pyrrolidinyl)methyl-[1,1']-biphen-4-yl]-1,4,5,6-tetrahydropyrazolo-[3,4-c]-pyridin-7-one (BMS-740808) a highly potent, selective, efficacious, and orally bioavailable inhibitor of blood coagulation factor Xa.

    PubMed

    Pinto, Donald J P; Orwat, Michael J; Quan, Mimi L; Han, Qi; Galemmo, Robert A; Amparo, Eugene; Wells, Brian; Ellis, Christopher; He, Ming Y; Alexander, Richard S; Rossi, Karen A; Smallwood, Angela; Wong, Pancras C; Luettgen, Joseph M; Rendina, Alan R; Knabb, Robert M; Mersinger, Lawrence; Kettner, Charles; Bai, Steven; He, Kan; Wexler, Ruth R; Lam, Patrick Y S

    2006-08-01

    Attempts to further optimize the pyrazole factor Xa inhibitors centered on masking the aryl aniline P4 moiety. Scaffold optimization resulted in the identification of a novel bicyclic pyrazolo-pyridinone scaffold which retained fXa potency. The novel bicyclic scaffold preserved all binding interactions observed with the monocyclic counterpart and importantly the carboxamido moiety was integrated within the scaffold making it less susceptible to hydrolysis. These efforts led to the identification of 1-[3-aminobenzisoxazol-5'-yl]-3-trifluoromethyl-6-[2'-(3-(R)-hydroxy-N-pyrrolidinyl)methyl-[1,1']-biphen-4-yl]-1,4,5,6-tetrahydropyrazolo-[3,4-c]-pyridin-7-one 6f (BMS-740808), a highly potent (fXa Ki=30 pM) with a rapid onset of inhibition (2.7x10(7) M-1 s-1) in vitro, selective (>1000-fold over other proteases), efficacious in the AVShunt thrombosis model, and orally bioavailable inhibitor of blood coagulation factor Xa.

  13. Blood flow controls coagulation onset via the positive feedback of factor VII activation by factor Xa

    PubMed Central

    2010-01-01

    Background Blood coagulation is a complex network of biochemical reactions, which is peculiar in that it is time- and space-dependent, and has to function in the presence of rapid flow. Recent experimental reports suggest that flow plays a significant role in its regulation. The objective of this study was to use systems biology techniques to investigate this regulation and to identify mechanisms creating a flow-dependent switch in the coagulation onset. Results Using a detailed mechanism-driven model of tissue factor (TF)-initiated thrombus formation in a two-dimensional channel we demonstrate that blood flow can regulate clotting onset in the model in a threshold-like manner, in agreement with existing experimental evidence. Sensitivity analysis reveals that this is achieved due to a combination of the positive feedback of TF-bound factor VII activation by activated factor X (Xa) and effective removal of factor Xa by flow from the activating patch depriving the feedback of "ignition". The level of this trigger (i.e. coagulation sensitivity to flow) is controlled by the activity of tissue factor pathway inhibitor. Conclusions This mechanism explains the difference between red and white thrombi observed in vivo at different shear rates. It can be speculated that this is a special switch protecting vascular system from uncontrolled formation and spreading of active coagulation factors in vessels with rapidly flowing blood. PMID:20102623

  14. [Management of major bleeding complications and emergency surgery in patients on long-term treatment with direct oral anticoagulants, thrombin or factor-Xa inhibitors. Proposals of the Working Group on Perioperative Haemostasis (GIHP) - March 2013].

    PubMed

    Pernod, G; Albaladejo, P; Godier, A; Samama, C M; Susen, S; Gruel, Y; Blais, N; Fontana, P; Cohen, A; Llau, J V; Rosencher, N; Schved, J F; de Maistre, E; Samama, M M; Mismetti, P; Sié, P

    2013-10-01

    New direct oral anticoagulants (NOAC), inhibitors of factor IIa or Xa, are expected to be widely used for the treatment of venous thromboembolic disease, or in case of atrial fibrillation. Such anticoagulant treatments are known to be associated with haemorrhagic complications. Moreover, it is likely that such patients on long-term treatment with NOAC will be exposed to emergency surgery or invasive procedures. Due to the present lack of experience in such conditions, we cannot make recommendations, but only propose management for optimal safety as regards the risk of bleeding in such emergency conditions. In this article, only dabigatran and rivaroxaban were discussed. For emergency surgery at risk of bleeding, we propose to dose the plasmatic concentration of drug. Levels inferior or equal to 30ng/mL for both dabigatran and rivaroxaban, should enable the realization of a high bleeding risk surgery. For higher concentration, it was proposed to postpone surgery by monitoring the evolution of the drug concentration. Action is then defined by the kind of NOAC and its concentration. If the dosage of the drug is not immediately available, proposals only based on the usual tests, PT and aPTT, also are presented. However, these tests do not really assess drug concentration or bleeding risk. In case of severe haemorrhage in a critical organ, it is proposed to reduce the effect of anticoagulant therapy using a nonspecific procoagulant drug (activated prothrombin concentrate, FEIBA, 30-50U/kg, or non-activated 4-factors prothrombin concentrates 50U/kg). For any other type of severe haemorrhage, the administration of such a procoagulant drug, potentially thrombogenic in these patients, will be discussed regarding concentration of NACO and possibilities for mechanical haemostasis.

  15. RUBY-1: a randomized, double-blind, placebo-controlled trial of the safety and tolerability of the novel oral factor Xa inhibitor darexaban (YM150) following acute coronary syndrome.

    PubMed

    Steg, Ph Gabriel; Mehta, Shamir R; Jukema, J Wouter; Lip, Gregory Y H; Gibson, C Michael; Kovar, Frantisek; Kala, Petr; Garcia-Hernandez, Alberto; Renfurm, Ronny W; Granger, Christopher B

    2011-10-01

    To establish the safety, tolerability and most promising regimen of darexaban (YM150), a novel, oral, direct factor Xa inhibitor, for prevention of ischaemic events in acute coronary syndrome (ACS). In a 26-week, multi-centre, double-blind, randomized, parallel-group study, 1279 patients with recent high-risk non-ST-segment or ST-segment elevation ACS received one of six darexaban regimens: 5 mg b.i.d., 10 mg o.d., 15 mg b.i.d., 30 mg o.d., 30 mg b.i.d., or 60 mg o.d. or placebo, on top of dual antiplatelet treatment. Primary outcome was incidence of major or clinically relevant non-major bleeding events. The main efficacy outcome was a composite of death, stroke, myocardial infarction, systemic thromboembolism, and severe recurrent ischaemia. Bleeding rates were numerically higher in all darexaban arms vs. placebo (pooled HR: 2.275; 95% CI: 1.13-4.60, P = 0.022). Using placebo as reference (bleeding rate 3.1%), there was a dose-response relationship (P = 0.009) for increased bleeding with increasing darexaban dose (6.2, 6.5, and 9.3% for 10, 30, and 60 mg daily, respectively), which was statistically significant for 30 mg b.i.d. (P = 0.002). There was no decrease (indeed a numerical increase in the 30 and 60 mg dose arms) in efficacy event rates with darexaban, but the study was underpowered for efficacy. Darexaban showed good tolerability without signs of liver toxicity. Darexaban when added to dual antiplatelet therapy after ACS produces an expected dose-related two- to four-fold increase in bleeding, with no other safety concerns but no signal of efficacy. Establishing the potential of low-dose darexaban in preventing major cardiac events after ACS requires a large phase III trial. ClinicalTrials.gov Identifier: NCT00994292.

  16. Management of major bleeding complications and emergency surgery in patients on long-term treatment with direct oral anticoagulants, thrombin or factor-Xa inhibitors: proposals of the working group on perioperative haemostasis (GIHP) - March 2013.

    PubMed

    Pernod, Gilles; Albaladejo, Pierre; Godier, Anne; Samama, Charles M; Susen, Sophie; Gruel, Yves; Blais, Normand; Fontana, Pierre; Cohen, Ariel; Llau, Juan V; Rosencher, Nadia; Schved, Jean-François; de Maistre, Emmanuel; Samama, Meyer M; Mismetti, Patrick; Sié, Pierre

    2013-01-01

    Direct new oral anticoagulants (NOACs) - inhibitors of thrombin or factor Xa - are intended to be used largely in the treatment of venous thromboembolic disease or the prevention of systematic embolism in atrial fibrillation, instead of vitamin K antagonists. Like any anticoagulant treatment, they are associated with spontaneous or provoked haemorrhagic risk. Furthermore, a significant proportion of treated patients are likely to be exposed to emergency surgery or invasive procedures. Given the absence of a specific antidote, the action to be taken in these situations must be defined. The lack of data means that it is only possible to issue proposals rather than recommendations, which will evolve according to accumulated experience. The proposals presented here apply to dabigatran (Pradaxa(®)) and rivaroxaban (Xarelto(®)); data for apixaban and edoxaban are still scarce. For urgent surgery with haemorrhagic risk, the drug plasma concentration should be less or equal to 30ng/mL for dabigatran and rivaroxaban should enable surgery associated with a high bleeding risk. Beyond that, if possible, the intervention should be postponed by monitoring the drug concentration. The course to follow is then defined according to the NOAC and its concentration. If the anticoagulant dosage is not immediately available, worse propositions, based on the usual tests (prothrombin time and activated partial thromboplastin time), are presented. However, these tests do not really assess drug concentration or the risk of bleeding that depends on it. In case of serious bleeding in a critical organ, the effect of anticoagulant therapy should be reduced using a non-specific procoagulant drug as a first-line approach: activated prothrombin complex concentrate (aPCC) (FEIBA(®) 30-50U/kg) or non-activated PCC (50U/kg). In addition, for any other type of severe haemorrhage, the administration of a procoagulant drug, which is potentially thrombogenic in these patients, is discussed according

  17. Future therapeutic directions for factor Xa inhibition in the prophylaxis and treatment of thrombotic disorders.

    PubMed

    Turpie, Alexander G G

    2003-11-15

    The targeted mechanism of factor Xa inhibition has been studied extensively, initially as prophylaxis for venous thromboembolism (VTE) in the orthopedic surgical setting. Future therapeutic directions for selective factor Xa inhibition in the management of other thrombotic diseases are discussed. Thromboembolic diseases can occur in the venous or arterial sides of the circulatory system. Factor Xa inhibition is a targeted approach to anticoagulation that resulted from significant advances in our understanding of the coagulation cascade. The factor Xa inhibitor fondaparinux has been studied extensively in the orthopedic surgical setting for the prophylaxis of VTE. Current investigations that are under way or completed evaluate the efficacy and safety of fondaparinux for the management of various thrombotic diseases. The future development of fondaparinux resides primarily in three therapeutic areas: prevention of VTE, treatment of VTE, and treatment of acute coronary syndromes. For the prevention of VTE, fondaparinux has been studied as extended prophylaxis following hip fracture surgery (PENTHIFRA Plus), for use in high-risk abdominal surgical patients (PEGASUS and APOLLO), and for use in medical patients (ARTEMIS). Studies evaluating fondaparinux for the treatment of VTE are part of the large MATISSE clinical program (MATISSE DVT and MATISSE PE). Fondaparinux was investigated in phase 2 studies for the treatment of acute coronary syndromes, including acute ST-segment myocardial infarction (PENTALYSE) and unstable angina (PENTUA). Encouraging data from these trials are the basis for phase 3 programs in this area (MICHELANGELO). The orthopedic prophylactic and nonorthopedic clinical programs for fondaparinux in the management of thrombosis support the concept that targeted inhibition of coagulation is an effective advance in antithrombotic therapy.

  18. Clinical decision-making for vitamin K-1 and K-2 deficiency and coronary artery calcification with warfarin therapy: are diet, factor Xa inhibitors or both the answer?

    PubMed

    Wahlqvist, Mark L; Tanaka, Kiyoshi; Tzeng, Bing-Hsiean

    2013-01-01

    Coronary artery calcification is a recognised risk factor for ischaemic heart disease and mortality. Evidence is now strong that Mönckeberg's arteriosclerosis, a form of vascular calcification, can be attributable to vitamin K deficiency, but that vitamin K-2, especially the MK-4 form from foods like cheese can be protective. Warfarin blocks the recycling of hepatic and peripheral vitamin K leading to secondary vitamin K deficiency with adverse effects on vasculature, bone, kidneys, brain and other tissues and systems (inflammatory, immune function and neoplasia at least). There is individual susceptibility to vitamin K deficiency and warfarin sensitivity, partly explicable in terms of genetic polymorphisms, epigenetics, diet and pharmacotherapy. The emergence of extensive coronary calcification in a man with atrial fibrillation treated for a decade with warfarin is described by way of illustration and to raise the present clinical management conundrums. Finally, a putative set of recommendations is provided.

  19. Specificity and selectivity profile of EP217609: a new neutralizable dual-action anticoagulant that targets thrombin and factor Xa

    PubMed Central

    Swanson, Richard; Petitou, Maurice

    2012-01-01

    EP217609 is a new dual-action parenteral anticoagulant that combines an indirect factor Xa inhibitor (fondaparinux analog) and a direct thrombin inhibitor (α-NAPAP analog) in a single molecule together with a biotin tag to allow avidin neutralization. EP217609 exhibits an unprecedented pharmacologic profile in showing high bioavailability, long plasma half-life, and potent antithrombotic activity in animals without the complications of thrombin rebound. Here we report the exceptional specificity and selectivity profile of EP217609. EP217609 inhibited thrombin with rapid kinetics (kon > 107M−1s−1), a high affinity (KI = 30-40pM), and more than 1000-fold selectivity over other coagulation and fibrinolytic protease targets, comparing favorably with the best direct thrombin inhibitors known. EP217609 bound antithrombin with high affinity (KD = 30nM) and activated the serpin to rapidly (kass ∼ 106M−1s−1) and selectively (> 20-fold) inhibit factor Xa. The dual inhibitor moieties of EP217609 acted largely independently with only modest linkage effects of ligand occupancy of one inhibitor moiety on the potency of the other (∼ 5-fold). In contrast, avidin binding effectively neutralized the potency of both inhibitor moieties (20- to 100-fold). These findings demonstrate the superior anticoagulant efficacy and rapid avidin neutralizability of EP217609 compared with anticoagulants that target thrombin or factor Xa alone. PMID:22144183

  20. 5-Chlorothiophene-2-carboxylic acid [(S)-2-[2-methyl-3-(2-oxopyrrolidin-1-yl)benzenesulfonylamino]-3-(4-methylpiperazin-1-yl)-3-oxopropyl]amide (SAR107375), a selective and potent orally active dual thrombin and factor Xa inhibitor.

    PubMed

    Meneyrol, Jerome; Follmann, Markus; Lassalle, Gilbert; Wehner, Volkmar; Barre, Guillaume; Rousseaux, Tristan; Altenburger, Jean-Michel; Petit, Frederic; Bocskei, Zsolt; Schreuder, Herman; Alet, Nathalie; Herault, Jean-Pascal; Millet, Laurence; Dol, Frederique; Florian, Peter; Schaeffer, Paul; Sadoun, Freddy; Klieber, Sylvie; Briot, Christophe; Bono, Françoise; Herbert, Jean-Marc

    2013-12-12

    Compound 15 (SAR107375), a novel potent dual thrombin and factor Xa inhibitor resulted from a rational optimization process. Starting from compound 14, with low factor Xa and modest anti-thrombin inhibitory activities (IC50's of 3.5 and 0.39 μM, respectively), both activities were considerably improved, notably through the incorporation of a neutral chlorothiophene P1 fragment and tuning of P2 and P3-P4 fragments. Final optimization of metabolic stability with microsomes led to the identification of 15, which displays strong activity in vitro vs factor Xa and thrombin (with Ki's of 1 and 8 nM, respectively). In addition 15 presents good selectivity versus related serine proteases (roughly 300-fold), including trypsin (1000-fold), and is very active (0.39 μM) in the thrombin generation time (TGT) coagulation assay in human platelet rich plasma (PRP). Potent in vivo activity in a rat model of venous thrombosis following iv and, more importantly, po administration was also observed (ED50 of 0.07 and 2.8 mg/kg, respectively). Bleeding liability was reduced in the rat wire coil model, more relevant to arterial thrombosis, with 15 (blood loss increase of 2-fold relative to the ED80 value) compared to rivaroxaban 2 and dabigatran etexilate 1a.

  1. Apixaban, an oral, direct, selective factor Xa inhibitor, in combination with antiplatelet therapy after acute coronary syndrome: results of the Apixaban for Prevention of Acute Ischemic and Safety Events (APPRAISE) trial.

    PubMed

    Alexander, John H; Becker, Richard C; Bhatt, Deepak L; Cools, Frank; Crea, Filippo; Dellborg, Mikael; Fox, Keith A A; Goodman, Shaun G; Harrington, Robert A; Huber, Kurt; Husted, Steen; Lewis, Basil S; Lopez-Sendon, Jose; Mohan, Puneet; Montalescot, Gilles; Ruda, Mikhail; Ruzyllo, Witold; Verheugt, Freek; Wallentin, Lars

    2009-06-09

    After an acute coronary syndrome, patients remain at risk of recurrent events. Apixaban, an oral direct factor Xa inhibitor, is a novel anticoagulant that may reduce these events but also poses a risk of bleeding. Apixaban for Prevention of Acute Ischemic and Safety Events (APPRAISE) was a phase 2, double-blind, placebo-controlled, dose-ranging study. Patients (n=1715) with recent ST-elevation or non-ST-elevation acute coronary syndrome were randomized to 6 months of placebo (n=611) or 1 of 4 doses of apixaban: 2.5 mg twice daily (n=317), 10 mg once daily (n=318), 10 mg twice daily (n=248), or 20 mg once daily (n=221). Nearly all patients received aspirin; 76% received clopidogrel. The primary outcome was International Society of Thrombosis and Hemostasis major or clinically relevant nonmajor bleeding. A secondary outcome was cardiovascular death, myocardial infarction, severe recurrent ischemia, or ischemic stroke. At the recommendation of the Data Monitoring Committee, the 2 higher-dose apixaban arms were discontinued because of excess total bleeding. Compared with placebo, apixaban 2.5 mg twice daily (hazard ratio, 1.78; 95% confidence interval, 0.91 to 3.48; P=0.09) and 10 mg once daily (hazard ratio, 2.45; 95% confidence interval, 1.31 to 4.61; P=0.005) resulted in a dose-dependent increase in major or clinically relevant nonmajor bleeding. Apixaban 2.5 mg twice daily (hazard ratio, 0.73; 95% confidence interval, 0.44 to 1.19; P=0.21) and 10 mg once daily (hazard ratio, 0.61; 95% confidence interval, 0.35 to 1.04; P=0.07) resulted in lower rates of ischemic events compared with placebo. The increase in bleeding was more pronounced and the reduction in ischemic events was less evident in patients taking aspirin plus clopidogrel than in those taking aspirin alone. We observed a dose-related increase in bleeding and a trend toward a reduction in ischemic events with the addition of apixaban to antiplatelet therapy in patients with recent acute coronary syndrome

  2. RUBY-1: a randomized, double-blind, placebo-controlled trial of the safety and tolerability of the novel oral factor Xa inhibitor darexaban (YM150) following acute coronary syndrome

    PubMed Central

    Steg, Ph. Gabriel; Mehta, Shamir R.; Jukema, J. Wouter; Lip, Gregory Y.H.; Gibson, C. Michael; Kovar, Frantisek; Kala, Petr; Garcia-Hernandez, Alberto; Renfurm, Ronny W.; Granger, Christopher B.

    2011-01-01

    Aims To establish the safety, tolerability and most promising regimen of darexaban (YM150), a novel, oral, direct factor Xa inhibitor, for prevention of ischaemic events in acute coronary syndrome (ACS). Methods In a 26-week, multi-centre, double-blind, randomized, parallel-group study, 1279 patients with recent high-risk non-ST-segment or ST-segment elevation ACS received one of six darexaban regimens: 5 mg b.i.d., 10 mg o.d., 15 mg b.i.d., 30 mg o.d., 30 mg b.i.d., or 60 mg o.d. or placebo, on top of dual antiplatelet treatment. Primary outcome was incidence of major or clinically relevant non-major bleeding events. The main efficacy outcome was a composite of death, stroke, myocardial infarction, systemic thromboembolism, and severe recurrent ischaemia. Results Bleeding rates were numerically higher in all darexaban arms vs. placebo (pooled HR: 2.275; 95% CI: 1.13–4.60, P = 0.022). Using placebo as reference (bleeding rate 3.1%), there was a dose–response relationship (P = 0.009) for increased bleeding with increasing darexaban dose (6.2, 6.5, and 9.3% for 10, 30, and 60 mg daily, respectively), which was statistically significant for 30 mg b.i.d. (P = 0.002). There was no decrease (indeed a numerical increase in the 30 and 60 mg dose arms) in efficacy event rates with darexaban, but the study was underpowered for efficacy. Darexaban showed good tolerability without signs of liver toxicity. Conclusions Darexaban when added to dual antiplatelet therapy after ACS produces an expected dose-related two- to four-fold increase in bleeding, with no other safety concerns but no signal of efficacy. Establishing the potential of low-dose darexaban in preventing major cardiac events after ACS requires a large phase III trial. ClinicalTrials.gov Identifier: NCT00994292 PMID:21878434

  3. The cost-effectiveness of oral direct factor Xa inhibitors compared with low-molecular-weight heparin for the prevention of venous thromboembolism prophylaxis in total hip or knee replacement surgery.

    PubMed

    Mahmoudi, Mandana; Sobieraj, Diana M

    2013-12-01

    Major orthopedic surgery is associated with a high risk of venous thromboembolism (VTE). Anticoagulants are recommended to prevent VTE, and recently an oral direct factor Xa inhibitor (FXaI) was approved for this indication. We compared the cost-effectiveness of FXaIs with low-molecular-weight heparin (LMWH) in patients undergoing total hip replacement (THR) or total knee replacement (TKR) surgery. A decision-tree model was developed to compare the cost-effectiveness of oral direct FXaIs (rivaroxaban, apixaban, and edoxaban) to subcutaneous LMWHs (enoxaparin and dalteparin), with separate models for THR and TKR. The analysis was conducted over a 180-day postoperative time horizon from the U.S. Medicare perspective. The model was developed using TreeAge Pro 2011 (TreeAge Software Inc., Williamstown, MA, USA). Efficacy and safety data (probabilities of distal and proximal deep vein thrombosis, symptomatic pulmonary embolism, and major bleeding) were derived from a systematic review and meta-analysis of phase II and III clinical trials. Costs and quality-adjusted life-years (QALYs) are reported. One-way and probabilistic sensitivity analyses were performed to evaluate parameter uncertainty. In the THR model, the average costs per patient for FXaIs and LMWHs were $18,762 and $18,897, respectively, and the QALYs were 0.938 and 0.932. In the TKR model, the average cost per patient for FXaIs and LMWHs were $18,804 and $18,991, respectively, and the QALYs were 0.935 and 0.931. In both models, FXaIs dominated LMWH (less costly and more efficacious). Neither model was sensitive to changes in any of the variables in the one-way sensitivity analyses. Probabilistic sensitivity analysis indicated that FXaIs were cost-effective in more than 99% of iterations in the THR population and in 98% of iterations in the TKR population assuming a willingness-to-pay threshold of $50,000/QALY. Oral direct FXaIs may be an economically dominant strategy compared with LMWHs for VTE prophylaxis

  4. Heparin chain-length dependence of factor Xa inhibition by antithrombin in plasma.

    PubMed

    Rezaie, Alireza R

    2007-01-01

    Heparin anticoagulants function by enhancing the inhibition of coagulation proteases by the serpin antithrombin (AT). A direct evaluation of the specific anti-factor Xa (fXa) activity of therapeutic heparins in the physiologically relevant plasma-based clotting assays has not been feasible since thrombin, the final protease of the cascade, is the primary target for inhibition by AT in the presence of heparin. To circumvent this problem, we developed an assay in which the native AT in plasma was replaced with an AT mutant which exhibits identical affinity for heparin and near normal reactivity for fXa, but does not react with thrombin and other coagulation proteases in either the absence or presence of heparin. This assay was used to distinguish the anti-fXa activity of different molecular weight heparins from their anti-thrombin activity in clotting assays which were initiated by the triggers of either the extrinsic or intrinsic coagulation pathway. The results suggest that the acceleration of fXa inhibition by AT exhibits a marked heparin chain-length dependence, with fondaparinux (a pentasaccharide) having the lowest and unfractionated heparin having the highest effect. Interestingly, comparative studies revealed that the fondaparinux-catalyzed acceleration of thrombin inhibition by AT also contributes to the prolongation of the clotting time, possibly suggesting that the anticoagulant function of the therapeutic pentasaccharide is mediated though the inhibition of both fXa and thrombin.

  5. The Tick-Derived Anticoagulant Madanin Is Processed by Thrombin and Factor Xa

    PubMed Central

    Figueiredo, Ana C.; de Sanctis, Daniele; Pereira, Pedro José Barbosa

    2013-01-01

    The cysteine-less peptidic anticoagulants madanin-1 and madanin-2 from the bush tick Haemaphysalis longicornis are the founding members of the MEROPS inhibitor family I53. It has been previously suggested that madanins exert their functional activity by competing with physiological substrates for binding to the positively charged exosite I (fibrinogen-binding exosite) of α-thrombin. We hereby demonstrate that competitive inhibition of α-thrombin by madanin-1 or madanin-2 involves binding to the enzyme's active site. Moreover, the blood coagulation factors IIa and Xa are shown to hydrolyze both inhibitors at different, although partially overlapping cleavage sites. Finally, the three-dimensional structure of the complex formed between human α-thrombin and a proteolytic fragment of madanin-1, determined by X-ray crystallography, elucidates the molecular details of madanin-1 recognition and processing by the proteinase. Taken together, the current findings establish the mechanism of action of madanins, natural anticoagulants that behave as cleavable competitive inhibitors of thrombin. PMID:23951260

  6. Coagulation-induced shedding of platelet glycoprotein VI mediated by factor Xa.

    PubMed

    Al-Tamimi, Mohammad; Grigoriadis, George; Tran, Huy; Paul, Eldho; Servadei, Patricia; Berndt, Michael C; Gardiner, Elizabeth E; Andrews, Robert K

    2011-04-07

    This study evaluated shedding of the platelet collagen receptor, glycoprotein VI (GPVI) in human plasma. Collagen or other ligands induce metalloproteinase-mediated GPVI ectodomain shedding, generating approximately 55-kDa soluble GPVI (sGPVI) and approximately 10-kDa platelet-associated fragments. In the absence of GPVI ligands, coagulation of platelet-rich plasma from healthy persons induced GPVI shedding, independent of added tissue factor, but inhibitable by metalloproteinase inhibitor, GM6001. Factor Xa (FXa) common to intrinsic and tissue factor-mediated coagulation pathways was critical for sGPVI release because (1) shedding was strongly blocked by the FXa-selective inhibitor rivaroxaban but not FIIa (thrombin) inhibitors dabigatran or hirudin; (2) Russell viper venom that directly activates FX generated sGPVI, with complete inhibition by enoxaparin (inhibits FXa and FIIa) but not hirudin; (3) impaired GPVI shedding during coagulation of washed platelets resuspended in FX-depleted plasma was restored by adding purified FX; and (4) purified FXa induced GM6001-inhibitable GPVI shedding from washed platelets. In 29 patients with disseminated intravascular coagulation, mean plasma sGPVI was 53.9 ng/mL (95% confidence interval, 39.9-72.8 ng/mL) compared with 12.5 ng/mL (95% confidence interval, 9.0-17.3 ng/mL) in thrombocytopenic controls (n = 36, P < .0001), and 14.6 ng/mL (95% confidence interval, 7.9-27.1 ng/mL) in healthy subjects (n = 25, P = .002). In conclusion, coagulation-induced GPVI shedding via FXa down-regulates GPVI under procoagulant conditions. FXa inhibitors have an unexpected role in preventing GPVI down-regulation.

  7. The relative molecular mass dependence of the anti-factor Xa properties of heparin.

    PubMed Central

    Ellis, V; Scully, M F; Kakkar, V V

    1986-01-01

    The effect of heparin fractions of various Mr, with high affinity for antithrombin III, on the kinetics of the reaction between factor Xa and antithrombin III have been studied using purified human proteins. Each of the heparin fractions, which varied between pentasaccharide and Mr 32,000, accelerated the inhibition of factor Xa although an increasing rate of inhibition was observed with increasing Mr. The chemically synthesized pentasaccharide preparation (Mr 1714) gave a maximum inhibition rate constant of 1.2 X 10(7) M-1 X min-1, compared with 6.3 X 10(4) M-1 X min-1 in the absence of heparin, and this rose progressively to 4.2 X 10(8) M-1 X min-1 with the two fractions of highest Mr (22,500 and 32,000). The 35-fold difference in inhibition rates observed with the high-affinity fractions was virtually abolished by the presence of 0.3 M-NaCl. The disparity in these rates of inhibition was shown to be due to a change in the Km for factor Xa when a two-substrate model of heparin catalysis was used. The Km for factor Xa rose from 28 nM for the fraction of Mr 32,000 to 770 nM for the pentasaccharide, whilst 0.3 M-NaCl also caused an increase in Km with the high-Mr fraction. These data suggest that the increased rates of inhibition observed with heparins of higher Mr may be due to an involvement of heparin binding to factor Xa as well as to antithrombin III. PMID:3800942

  8. Cleavage of spike protein of SARS coronavirus by protease factor Xa is associated with viral infectivity

    SciTech Connect

    Du, Lanying; Kao, Richard Y.; Zhou, Yusen; He, Yuxian; Zhao, Guangyu; Wong, Charlotte; Jiang, Shibo; Yuen, Kwok-Yung; Jin, Dong-Yan; Zheng, Bo-Jian . E-mail: bzheng@hkucc.hku.hk

    2007-07-20

    The spike (S) protein of SARS coronavirus (SARS-CoV) has been known to recognize and bind to host receptors, whose conformational changes then facilitate fusion between the viral envelope and host cell membrane, leading to viral entry into target cells. However, other functions of SARS-CoV S protein such as proteolytic cleavage and its implications to viral infection are incompletely understood. In this study, we demonstrated that the infection of SARS-CoV and a pseudovirus bearing the S protein of SARS-CoV was inhibited by a protease inhibitor Ben-HCl. Also, the protease Factor Xa, a target of Ben-HCl abundantly expressed in infected cells, was able to cleave the recombinant and pseudoviral S protein into S1 and S2 subunits, and the cleavage was inhibited by Ben-HCl. Furthermore, this cleavage correlated with the infectivity of the pseudovirus. Taken together, our study suggests a plausible mechanism by which SARS-CoV cleaves its S protein to facilitate viral infection.

  9. [Anti-factor Xa activity of enoxaparin for thromboprophylaxis in nonsurgical patients is dependent on body mass].

    PubMed

    Jiménez, David; Díaz, Gema; Iglesias, Ana; César, Jesús; García-Avello, Angel; Martí, David; Escobar, Carlos; Vidal, Rafael; Sueiro, Antonio

    2008-12-01

    Thromboprophylaxis with a fixed dose of low-molecular-weight heparin is recommended for hospitalized acutely ill medical patients. The purpose of this study was to assess whether the anti-factor Xa (anti-Xa) activity of enoxaparin prescribed for venous thromboembolism prophylaxis depends on body mass index (BMI) in patients hospitalized for an acute respiratory disease. All patients admitted to the respiratory medicine department (January-December 2006) for an acute respiratory disease, and for whom pharmacologic thromboprophylaxis was indicated, were included in the study. Anti-Xa activity was measured 4 hours after administration of enoxaparin on the third day of hospitalization. The primary outcome was anti-Xa activity in relation to BMI. One hundred twelve patients were enrolled. Mean anti-Xa activity decreased with each BMI quartile (0.28, 0.23, 0.15, and 0.13 U/mL for quartiles 1, 2, 3, and 4, respectively). In the multivariate analysis, BMI was the only predictor of inadequate anti-Xa activity (odds ratio, 1.14; 95% confidence interval, 10.5-1.24; P< .002) after adjustment for age, sex, and serum creatinine levels. Two episodes of symptomatic proximal deep vein thrombosis were diagnosed in the month after hospitalization; both occurred in patients who had inadequate anti-Xa activity. Anti-Xa activity is dependent on BMI in hospitalized acute medical patients receiving enoxaparin for thromboprophylaxis.

  10. Stabilizing of a globular protein by a highly complex water network: a molecular dynamics simulation study on factor Xa.

    PubMed

    Wallnoefer, Hannes G; Handschuh, Sandra; Liedl, Klaus R; Fox, Thomas

    2010-06-03

    The role of water molecules is increasingly attracting attention in structural biology, and many studies have demonstrated their crucial contribution to the stability and function of proteins. Here, we present molecular dynamics studies on factor Xa (fXa) to investigate the effect of water molecules in this serine protease. fXa is a key enzyme in the blood coagulation cascade, and thus, an important target for antithrombotic drugs. A reasonable representation of the structure is crucial for an investigation at the molecular level and, thus, a prerequisite for structure-based drug design. Simulations of well-resolved fXa X-ray structures with different sets of water molecules show the importance of a well-determined water set for the simulation. We discuss implications of different water sets on the structure and dynamics of fXa.

  11. Annexin A2 contributes to lung injury and fibrosis by augmenting factor Xa fibrogenic activity.

    PubMed

    Schuliga, Michael; Jaffar, Jade; Berhan, Asres; Langenbach, Shenna; Harris, Trudi; Waters, David; Lee, Peter V S; Grainge, Christopher; Westall, Glen; Knight, Darryl; Stewart, Alastair G

    2017-05-01

    In lung injury and disease, including idiopathic pulmonary fibrosis (IPF), extravascular factor X is converted into factor Xa (FXa), a coagulant protease with fibrogenic actions. Extracellular annexin A2 binds to FXa, augmenting activation of the protease-activated receptor-1 (PAR-1). In this study, the contribution of annexin A2 in lung injury and fibrosis was investigated. Annexin A2 immunoreactivity was observed in regions of fibrosis, including those associated with fibroblasts in lung tissue of IPF patients. Furthermore, annexin A2 was detected in the conditioned media and an EGTA membrane wash of human lung fibroblast (LF) cultures. Incubation with human plasma (5% vol/vol) or purified FXa (15-50 nM) evoked fibrogenic responses in LF cultures, with FXa increasing interleukin-6 (IL-6) production and cell number by 270 and 46%, respectively (P < 0.05, n = 5-8). The fibrogenic actions of plasma or FXa were attenuated by the selective FXa inhibitor apixaban (10 μM, or antibodies raised against annexin A2 or PAR-1 (2 μg/ml). FXa-stimulated LFs from IPF patients (n = 6) produced twice as much IL-6 as controls (n = 10) (P < 0.05), corresponding with increased levels of extracellular annexin A2. Annexin A2 gene deletion in mice reduced bleomycin-induced increases in bronchoalveolar lavage fluid (BALF) IL-6 levels and cell number (*P < 0.05; n = 4-12). Lung fibrogenic gene expression and dry weight were reduced by annexin A2 gene deletion, but lung levels of collagen were not. Our data suggest that annexin A2 contributes to lung injury and fibrotic disease by mediating the fibrogenic actions of FXa. Extracellular annexin A2 is a potential target for the treatment of IPF. Copyright © 2017 the American Physiological Society.

  12. Cerebral Venous Thromboembolism in Antiphospholipid Syndrome Successfully Treated with the Combined Use of an Anti-Xa Inhibitor and Corticosteroid.

    PubMed

    Sugie, Masayuki; Iizuka, Natsuko; Shimizu, Yuki; Ichikawa, Hiroo

    2015-01-01

    We herein report a case presenting with cerebral venous sinus thrombosis (CVST) associated with primary antiphospholipid syndrome (APS). The patient developed recurrent CVST followed by a hemorrhagic ischemic stroke despite the use of warfarin during the appropriate therapeutic window. Thus, we substituted warfarin to rivaroxaban with prednisolone and obtained a good clinical course. In addition to the effect of prednisolone of inhibiting elevated lupus anticoagulants and the recurrence of arterial thrombosis, rivaroxaban may prevent CVST and inhibit hypercoagulability induced by corticosteroids. The combination of an anti-Xa inhibitor and corticosteroid may be an alternative treatment for CVST and arterial thrombus with warfarin-resistant APS.

  13. Functional assembly of intrinsic coagulation proteases on monocytes and platelets. Comparison between cofactor activities induced by thrombin and factor Xa

    PubMed Central

    1992-01-01

    Generation of coagulation factor Xa by the intrinsic pathway protease complex is essential for normal activation of the coagulation cascade in vivo. Monocytes and platelets provide membrane sites for assembly of components of this protease complex, factors IXa and VIII. Under biologically relevant conditions, expression of functional activity by this complex is associated with activation of factor VIII to VIIIa. In the present studies, autocatalytic regulatory pathways operating on monocyte and platelet membranes were investigated by comparing the cofactor function of thrombin-activated factor VIII to that of factor Xa-activated factor VIII. Reciprocal functional titrations with purified human factor VIII and factor IXa were performed at fixed concentrations of human monocytes, CaCl2, factor X, and either factor IXa or factor VIII. Factor VIII was preactivated with either thrombin or factor Xa, and reactions were initiated by addition of factor X. Rates of factor X activation were measured using chromogenic substrate specific for factor Xa. The K1/2 values, i.e., concentration of factor VIIIa at which rates were half maximal, were 0.96 nM with thrombin- activated factor VIII and 1.1 nM with factor Xa-activated factor VIII. These values are close to factor VIII concentration in plasma. The Vsat, i.e., rates at saturating concentrations of factor VIII, were 33.3 and 13.6 nM factor Xa/min, respectively. The K1/2 and Vsat values obtained in titrations with factor IXa were not significantly different from those obtained with factor VIII. In titrations with factor X, the values of Michaelis-Menten coefficients (Km) were 31.7 nM with thrombin- activated factor VIII, and 14.2 nM with factor Xa-activated factor VIII. Maximal rates were 23.4 and 4.9 nM factor Xa/min, respectively. The apparent catalytic efficiency was similar with either form of factor VIIIa. Kinetic profiles obtained with platelets as a source of membrane were comparable to those obtained with monocytes

  14. Discovery of a factor Xa inhibitor (3R,4R)-1-(2,2-difluoro-ethyl)-pyrrolidine-3,4-dicarboxylic acid 3-[(5-chloro-pyridin-2-yl)-amide] 4-[[2-fluoro-4-(2-oxo-2H-pyridin-1-yl)-phenyl]-amide] as a clinical candidate.

    PubMed

    Anselm, Lilli; Banner, David W; Benz, Jörg; Zbinden, Katrin Groebke; Himber, Jacques; Hilpert, Hans; Huber, Walter; Kuhn, Bernd; Mary, Jean-Luc; Otteneder, Michael B; Panday, Narendra; Ricklin, Fabienne; Stahl, Martin; Thomi, Stefan; Haap, Wolfgang

    2010-09-01

    A series of (3R,4R)-pyrrolidine-3,4-dicarboxylic acid amides was investigated with respect to their factor Xa inhibitory activity, selectivity, pharmacokinetic properties, and ex vivo antithrombotic activity. The clinical candidate from this series, R1663, exhibits excellent selectivity against a panel of serine proteases and good pharmacokinetic properties in rats and monkeys. A Phase I clinical study with R1663 has been finalized. Copyright 2010 Elsevier Ltd. All rights reserved.

  15. Discovery of 1-(4-Methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro- 1H-pyrazolo[3,4-c]pyridine-3-carboxamide (Apixaban, BMS-562247), a Highly Potent, Selective, Efficacious, and Orally Bioavailable Inhibitor of Blood Coagulation Factor Xa

    SciTech Connect

    Pinto, Donald J.P.; Orwat, Michael J.; Koch, Stephanie; Rossi, Karen A.; Alexander, Richard S.; Smallwood, Angela; Wong, Pancras C.; Rendina, Alan R.; Luettgen, Joseph M.; Knabb, Robert M.; He, Kan; Xin, Baomin; Wexler, Ruth R.; Lam, Patrick Y.S.

    2010-03-08

    Efforts to identify a suitable follow-on compound to razaxaban (compound 4) focused on modification of the carboxamido linker to eliminate potential in vivo hydrolysis to a primary aniline. Cyclization of the carboxamido linker to the novel bicyclic tetrahydropyrazolopyridinone scaffold retained the potent fXa binding activity. Exceptional potency of the series prompted an investigation of the neutral P{sub 1} moieties that resulted in the identification of the p-methoxyphenyl P{sub 1}, which retained factor Xa binding affinity and good oral bioavailability. Further optimization of the C-3 pyrazole position and replacement of the terminal P{sub 4} ring with a neutral heterocycle culminated in the discovery of 1-(4-methoxyphenyl)-7-oxo-6-(4-(2-oxopiperidin-1-yl)phenyl)-4,5,6,7-tetrahydro-1H-pyrazolo[3,4-c]pyridine-3-carboxamide (apixaban, compound 40). Compound 40 exhibits a high degree of fXa potency, selectivity, and efficacy and has an improved pharmacokinetic profile relative to 4.

  16. Acylcarnitines are anticoagulants that inhibit factor Xa and are reduced in venous thrombosis, based on metabolomics data

    PubMed Central

    Deguchi, Hiroshi; Banerjee, Yajnavalka; Trauger, Sunia; Siuzdak, Gary; Kalisiak, Ewa; Fernández, José A.; Hoang, Linh; Tran, Minerva; Yegneswaran, Subramanian; Elias, Darlene J.

    2015-01-01

    In many patients with deep vein thrombosis and pulmonary embolism (venous thromboembolism, VTE), biomarkers or genetic risk factors have not been identified. To discover novel plasma metabolites associated with VTE risk, we employed liquid chromatography-mass spectrometry-based untargeted metabolomics, which do not target any specific metabolites. Using the Scripps Venous Thrombosis Registry population for a case-control study, we discovered that 10:1 and 16:1 acylcarnitines were low in plasmas of the VTE patient group compared with matched controls, respectively. Data from targeted metabolomics studies showed that several long-chain acylcarnitines (10:1, 12:0, 12:2, 18:1, and 18:2) were lower in the VTE group. Clotting assays were used to evaluate a causal relationship for low acylcarnitines in patients with VTE. Various acylcarnitines inhibited factor Xa-initiated clotting. Inhibition of factor Xa by acylcarnitines was greater for longer acyl chains. Mechanistic studies showed that 16:0 acylcarnitine had anticoagulant activity in the absence of factor Va or phospholipids. Surface plasmon resonance investigations revealed that 16:0 acylcarnitine was bound to factor Xa and that binding did not require the γ-carboxy glutamic acid domain. In summary, our study identified low plasma levels of acylcarnitines in patients with VTE and showed that acylcarnitines have anticoagulant activity related to an ability to bind and inhibit factor Xa. PMID:26175037

  17. Laser-induced lanthanide luminescence as a probe of metal ion-binding sites of human Factor Xa.

    PubMed

    Rhee, M J; Horrocks, W D; Kosow, D P

    1984-06-25

    7F0 ---- 5D0 excitation spectroscopy of Eu(III) has shown that human Factor Xa has two high affinity lanthanide ion-binding sites. The deuterium isotope effect on the reciprocal lifetime (tau-1) of excited Eu(III) in human Factor Xa has indicated that 2 to 3 water molecules remain on Eu(III) after being complexed by Factor Xa, suggesting that 3-6 ligand atoms are provided by the protein, probably through two or three gamma-carboxyglutamic acids (GLA). F orster -type interlanthanide energy transfer has been utilized to measure the distance between the high affinity metal ion-binding sites of human Factor Xa using Tb(III) as an energy donor and Nd(III), Ho(III), or Er(III) as energy acceptors. Tau-1 values of Tb(III) in the presence of the acceptor ions Nd(III), Ho(III), and Er(III) were 1.90, 1.66, and 1.76 ms-1, respectively, which compared to 1.31 ms-1 in the presence of the nonacceptor ion Gd(III), yield energy transfer efficiencies of 0.29, 0.20, and 0.24, respectively. From these efficiencies and published critical distances (R0) ( Horrocks , W. DeW ., Jr., Rhee , M-J., Snyder, A. P., and Sudnick , D. R. (1980) J. Am. Chem. Soc. 102, 3650-3652), the distance between two high affinity sites is estimated to be 10.7 A. Based on these data, we propose that the two high affinity sites of human Factor Xa consist of two paired GLA residues; GLA-19, GLA-20 and GLA-25, GLA-26 together with one of the remaining single GLA residues for each site.

  18. A novel microfluidic anti-factor Xa assay device for monitoring anticoagulant therapy at the point-of-care

    NASA Astrophysics Data System (ADS)

    Harris, Leanne F.; Rainey, Paul; Castro-López, Vanessa; O'Donnell, James S.; Killard, Anthony J.

    2013-05-01

    Millions of patients worldwide are receiving anticoagulant therapy to treat hypercoagulable diseases. While standard testing is still performed in the central laboratory, point-of-care (POC) diagnostics are being developed due to the increasing number of patients requiring long-term anticoagulation and with a need for more personalized and targeted therapy. Many POC devices on the market focus on clot measurement, a technique which is limited in terms of variability, highlighting the need for more reliable assays of anticoagulant status. The anti-Xa assay, a factor specific optical assay, was developed to measure the extent to which exogenous factor Xa (FXa) is inhibited by heparinantithrombin complexes. We have developed a novel microfluidic device and assay for monitoring the effect of heparin anticoagulant therapy at the point-of-care. The assay which was also developed in our institute is based on the anti-Xa assay principle but uses fluorescence as the method of detection. Our device is a disposable laminate microfluidic strip, fabricated from the cyclic polyolefin (COP), Zeonor®, which is extremely suitable for application to fluorescent device platforms. We present data on the execution of the anti-Xa assay in this microfluidic format, demonstrating that the assay can be used to measure heparin in human plasma samples from 0 to 0.8 U/ml, with average assay reproducibility of 8% and a rapid result obtained within 60 seconds. Results indicate that with further development, the fluorogenic anti-Xa assay and device could become a successful method for monitoring anticoagulant therapy.

  19. Coagulation factor X inhibitor from hundred-pace snake (Deinagkistrodon acutus) venom.

    PubMed

    Cox, A C

    1993-11-01

    Deinagkistrodon acutus venom contains a collection of anticoagulant proteins that has been reported to prevent prothrombinase assembly (Teng and Seegers, 1981, Thromb. Res. 23, 255). A partial sequence indicates that these proteins are related to the functionally equivalent protein in Trimeresurus flavoviridis (Atoda et al., 1991, J. Biochem. 106, 808). Inhibition of prothrombinase, the complex of Factors Xa and Va combined with phospholipids, is expressed in bovine, human, and rat plasmas as indicated by an assay dependent on only prothrombinase activity. The concentration dependence of inhibition of prothrombin conversion by different combinations of the components of bovine prothrombinase under the same conditions yielded estimates of apparent dissociation constants of 104 nM and 2 nM for complexes of the inhibitor with Factor Xa and with Factors Xa and Va, respectively. Because this inhibitor does not prevent Factor Xa alone from converting prothrombin, but blocks the other combinations, we conclude the inhibitor prevents the complex of Factors Xa and Va from binding to phospholipid surfaces and to prothrombin. The inhibitor also blocks the activation of Factor X by Factor VIIa and thromboplastin as well. However, the inhibitor has no effect on thrombin-induced clotting or fibrinolysis induced by either plasminogen activator or streptokinase. Therefore, this inhibitor has several properties required of an anticoagulant, therapeutic agent.

  20. Group D prothrombin activators from snake venom are structural homologues of mammalian blood coagulation factor Xa.

    PubMed Central

    Rao, Veena S; Joseph, Jeremiah S; Kini, R Manjunatha

    2003-01-01

    Procoagulant venoms of several Australian elapids contain proteinases that specifically activate prothrombin; among these, Group D activators are functionally similar to coagulation factor Xa (FXa). Structural information on this class of prothrombin activators will contribute significantly towards understanding the mechanism of FXa-mediated prothrombin activation. Here we present the purification of Group D prothrombin activators from three Australian snake venoms (Hoplocephalus stephensi, Notechis scutatus scutatus and Notechis ater niger) using a single-step method, and their N-terminal sequences. The N-terminal sequence of the heavy chain of hopsarin D (H. stephensi) revealed that a fully conserved Cys-7 was substituted with a Ser residue. We therefore determined the complete amino acid sequence of hopsarin D. Hopsarin D shows approximately 70% similarity with FXa and approximately 98% similarity with trocarin D, a Group D prothrombin activator from Tropidechis carinatus. It possesses the characteristic Gla domain, two epidermal growth factor-like domains and a serine proteinase domain. All residues important for catalysis are conserved, as are most regions involved in interactions with factor Va and prothrombin. However, there are some structural differences. Unlike FXa, hopsarin D is glycosylated in both its chains: in light-chain residue 52 and heavy-chain residue 45. The glycosylation on the heavy chain is a large carbohydrate moiety adjacent to the active-site pocket. Overall, hopsarin D is structurally and functionally similar to mammalian coagulation FXa. PMID:12403650

  1. Hysteresis-like binding of coagulation factors X/Xa to procoagulant activated platelets and phospholipids results from multistep association and membrane-dependent multimerization.

    PubMed

    Podoplelova, Nadezhda A; Sveshnikova, Anastasia N; Kurasawa, James H; Sarafanov, Andrey G; Chambost, Herve; Vasil'ev, Sergey A; Demina, Irina A; Ataullakhanov, Fazly I; Alessi, Marie-Christine; Panteleev, Mikhail A

    2016-06-01

    Binding of coagulation factors X (fX) and Xa (fXa) to activated platelets is required for the formation of membrane-dependent enzymatic complexes of intrinsic tenase and prothrombinase. We carried out an in-depth characterization of fX/fXa binding to phospholipids and gel-filtered, thrombin-activated platelets. Flow cytometry, surface plasmon resonance, and computational modeling were used to investigate interactions of fX/fXa with the membranes. Confocal microscopy was employed to study fXa binding to platelet thrombi formed in flowing whole blood under arterial conditions. Binding of fX/fXa to either vesicles or procoagulant platelets did not follow a traditional one-step reversible binding model. Their dissociation was a two-step process resulting in a plateau that was up to 10-fold greater than the saturation value observed in the association experiments. Computational modeling and experimental evidence suggested that this was caused by a combination of two-step association (mainly for fX) and multimerization on the membrane (mainly for fXa). Importantly, fX formed multimers with fXa, thereby improving its retention. The same binding/dissociation hysteresis was observed for annexin V known to form trimers on the membranes. Experiments with platelets from gray syndrome patients showed that alpha-granular factor Va provided an additional high-affinity binding site for fXa that did not affect the hysteresis. Confocal microscopy observation of fXa binding to platelet thrombi in a flow chamber and its wash-out confirmed that this phenomenon persisted under physiologically relevant conditions. This suggests its possible role of "locking" coagulation factors on the membrane and preventing their inhibition in plasma and removal from thrombi by flow.

  2. Protein-Templated Formation of an Inhibitor of the Blood Coagulation Factor Xa through a Background-Free Amidation Reaction.

    PubMed

    Jaegle, Mike; Steinmetzer, Torsten; Rademann, Jörg

    2017-03-20

    Protein-templated reactions enable the target-guided formation of protein ligands from reactive fragments, ideally with no background reaction. Herein, we investigate the templated formation of amides. A nucleophilic fragment that binds to the coagulation factor Xa was incubated with the protein and thirteen differentially activated dipeptides. The protein induced a non-catalytic templated reaction for the phenyl and trifluoroethyl esters; the latter was shown to be a completely background-free reaction. Starting from two fragments with millimolar affinity, a 29 nm superadditive inhibitor of factor Xa was obtained. The fragment ligation reaction was detected with high sensitivity by an enzyme activity assay and by mass spectrometry. The reaction progress and autoinhibition of the templated reaction by the formed ligation product were determined, and the structure of the protein-inhibitor complex was elucidated.

  3. Protein‐Templated Formation of an Inhibitor of the Blood Coagulation Factor Xa through a Background‐Free Amidation Reaction

    PubMed Central

    Jaegle, Mike; Steinmetzer, Torsten

    2017-01-01

    Abstract Protein‐templated reactions enable the target‐guided formation of protein ligands from reactive fragments, ideally with no background reaction. Herein, we investigate the templated formation of amides. A nucleophilic fragment that binds to the coagulation factor Xa was incubated with the protein and thirteen differentially activated dipeptides. The protein induced a non‐catalytic templated reaction for the phenyl and trifluoroethyl esters; the latter was shown to be a completely background‐free reaction. Starting from two fragments with millimolar affinity, a 29 nm superadditive inhibitor of factor Xa was obtained. The fragment ligation reaction was detected with high sensitivity by an enzyme activity assay and by mass spectrometry. The reaction progress and autoinhibition of the templated reaction by the formed ligation product were determined, and the structure of the protein–inhibitor complex was elucidated. PMID:28199769

  4. The effect of Ca2+, phospholipid and factor V on the anti-(factor Xa) activity of heparin and its high-affinity oligosaccharides.

    PubMed Central

    Barrowcliffe, T W; Havercroft, S J; Kemball-Cook, G; Lindahl, U

    1987-01-01

    The influence of Ca2+, phospholipid and Factor V was determined on the rate of inactivation of Factor Xa by antithrombin III, in the absence and in the presence of unfractionated heparin and of three high-affinity heparin oligosaccharides in the Mr range 1500-6000. In the absence of heparin the addition of Ca2+, phospholipid and Factor V caused a 4-fold decrease in rate of inactivation of Factor Xa. As concentrations of unfractionated heparin were increased the protective effect of Ca2+/phospholipid/Factor V was gradually abolished, and at a concentration of 2.4 nM there were no differences in rates of neutralization of Factor Xa in the presence or absence of Ca2+, phospholipid and Factor V. In contrast, heparin decasaccharide (Mr 3000) and pentasaccharide (Mr 1500) fragments were unable to overcome the protective effect of Ca2+/phospholipid/Factor V; in the presence of these components their catalytic efficiencies were 16-fold and 40-fold less respectively than that of unfractionated heparin. A heparin 20-22-saccharide fragment (Mr approx. 6000) gave similar inactivation rates in the presence and in the absence of Ca2+/phospholipid/Factor V. Human and bovine Factor Xa gave similar results. These results indicate that in the presence of Ca2+/phospholipid/Factor V optimum inhibition of Factor Xa requires a saccharide sequence of heparin additional to that involved in binding to antithrombin III. The use of free enzyme for the assessment of anti-(Factor Xa) activity of low-Mr heparin fractions could give misleading results. PMID:3606581

  5. Novel direct factor Xa inhibitory compounds from Tenebrio molitor with anti-platelet aggregation activity.

    PubMed

    Lee, Wonhwa; Kim, Mi-Ae; Park, InWha; Hwang, Jae Sam; Na, MinKyun; Bae, Jong-Sup

    2017-08-24

    Tenebrio molitor is an edible insect that has antimicrobial, anticancer, and antihypertensive effects. The aim of this study was to identify the unreported bioactive compounds from T. molitor larvae with inhibitory activities against factor Xa (FXa) and platelet aggregation. Isolated compounds were evaluated for their anti-FXa and anti-platelet aggregation properties by monitoring clotting time, platelet aggregation, FXa activity, and thrombus formation. A diketopiperazine (1, cyclo(L-Pro-L-Tyr)) and a phenylethanoid (2, N-acetyltyramine) were isolated and inhibited the catalytic activity of FXa in a mixed inhibition model and inhibited platelet aggregation induced by adenosine diphosphate (ADP) and U46619. They inhibited ADP- and U46619-induced phosphorylation of myristoylated alanine-rich C kinase substrate (MARCKS) and the expression of P-selectin and PAC-1 in platelets. They also improved the production of nitric oxide and inhibited the oversecretion of endothelin-1 compared to that of the ADP- or U46619-treated group. In an animal model of arterial and pulmonary thrombosis, the isolated compounds showed enhanced antithrombotic effects. They also elicited anticoagulant effects in mice. Compounds 1-2 inhibited ADP-, collagen-, or U46619-induced platelet aggregation and showed similar anti-thrombotic efficacy to rivaroxaban, a positive control. Therefore, 1-2 could serve as candidates and provide scaffolds for the development of new anti-FXa and anti-platelet drugs. Copyright © 2017 Elsevier Ltd. All rights reserved.

  6. Effects of factor Xa on the expression of proteins in femoral arteries from type 2 diabetic patients.

    PubMed

    López-Farré, Antonio J; Rodriguez-Sierra, Pablo; Modrego, Javier; Segura, Antonio; Martín-Palacios, Naiara; Saiz, Ana M; Zamorano-León, José J; Duarte, Juan; Serrano, Javier; Moñux, Guillermo

    2014-12-01

    Further to its pivotal role in haemostasis, factor Xa (FXa) promotes effects on the vascular wall. The purpose of the study was to evaluate if FXa modifies the expression level of energy metabolism and oxidative stress-related proteins in femoral arteries obtained from type 2 diabetic patients with end-stage vasculopathy. Femoral arteries were obtained from 12 type 2 diabetic patients who underwent leg amputation. Segments from the femoral arteries were incubated in vitro alone and in the presence of 25 nmol l(-1) FXa and 25 nmol l(-1) FXa + 50 nmol l(-1) rivaroxaban. In the femoral arteries, FXa increased triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase isotype 1 expression but decreased pyruvate dehydrogenase expression. These facts were accompanied by an increased content of acetyl-CoA. Aconitase activity was reduced in FXa-incubated femoral arteries as compared with control. Moreover, FXa increased the protein expression level of oxidative stress-related proteins which was accompanied by an increased malonyldialdehyde arterial content. The FXa inhibitor, rivaroxaban, failed to prevent the reduced expression of pyruvate dehydrogenase induced by FXa but reduced acetyl-CoA content and reverted the decreased aconitase activity observed with FXa alone. Rivaroxaban + FXa but not FXa alone increased the expression level of carnitine palmitoyltransferase I and II, two mitochondrial long chain fatty acid transporters. Rivaroxaban also prevented the increased expression of oxidative stress-related proteins induced by FXa alone. In femoral isolated arteries from type 2 diabetic patients with end-stage vasculopathy, FXa promoted disruption of the aerobic mitochondrial metabolism. Rivaroxaban prevented such effects and even seemed to favour long chain fatty acid transport into mitochondria. © 2014 The British Pharmacological Society.

  7. Effects of factor Xa on the expression of proteins in femoral arteries from type 2 diabetic patients

    PubMed Central

    López-Farré, Antonio J; Rodriguez-Sierra, Pablo; Modrego, Javier; Segura, Antonio; Martín-Palacios, Naiara; Saiz, Ana M; Zamorano-León, José J; Duarte, Juan; Serrano, Javier; Moñux, Guillermo

    2014-01-01

    Aim Further to its pivotal role in haemostasis, factor Xa (FXa) promotes effects on the vascular wall. The purpose of the study was to evaluate if FXa modifies the expression level of energy metabolism and oxidative stress-related proteins in femoral arteries obtained from type 2 diabetic patients with end-stage vasculopathy. Methods Femoral arteries were obtained from 12 type 2 diabetic patients who underwent leg amputation. Segments from the femoral arteries were incubated in vitro alone and in the presence of 25 nmol l−1 FXa and 25 nmol l−1 FXa + 50 nmol l−1 rivaroxaban. Results In the femoral arteries, FXa increased triosephosphate isomerase and glyceraldehyde-3-phosphate dehydrogenase isotype 1 expression but decreased pyruvate dehydrogenase expression. These facts were accompanied by an increased content of acetyl-CoA. Aconitase activity was reduced in FXa-incubated femoral arteries as compared with control. Moreover, FXa increased the protein expression level of oxidative stress-related proteins which was accompanied by an increased malonyldialdehyde arterial content. The FXa inhibitor, rivaroxaban, failed to prevent the reduced expression of pyruvate dehydrogenase induced by FXa but reduced acetyl-CoA content and reverted the decreased aconitase activity observed with FXa alone. Rivaroxaban + FXa but not FXa alone increased the expression level of carnitine palmitoyltransferase I and II, two mitochondrial long chain fatty acid transporters. Rivaroxaban also prevented the increased expression of oxidative stress-related proteins induced by FXa alone. Conclusions In femoral isolated arteries from type 2 diabetic patients with end-stage vasculopathy, FXa promoted disruption of the aerobic mitochondrial metabolism. Rivaroxaban prevented such effects and even seemed to favour long chain fatty acid transport into mitochondria. PMID:25041869

  8. Deuterium Solvent Isotope Effect and Proton Inventory Studies of Factor Xa-catalyzed Reactions†

    PubMed Central

    Zhang, Daoning; Kovach, Ildiko M.

    2008-01-01

    Kinetic solvent isotope effects (KSIE) for the Factor Xa (FXa)-catalyzed activation of prothrombin in the presence and absence of Factor Va (FVa) and 5.0 ×10−5 M phospholipid vesicles, are slightly inverse, 0.82−0.93, when substrate concentrations are at 0.2 Km. This is consistent with rate-determining association of the enzyme-prothrombin assembly, rather than rate-limiting chemical transformation. FVa is known to effect a major conformational change to expose the first scissile bond in prothrombin, which is the likely event triggering a major solvent rearrangement. At prothrombin concentrations >5 Km the KSIE is 1.6 ± 0.3, when FXa is in 1 : 1 ratio with FVa, but becomes increasingly inverse, 0.30 ± 0.05 and 0.19 ± 0.04, when FXa : FVa 1:4, with increasing FXa concentration and substrate concentration. The rate-determining step changes with the conditions, but the chemical step is not limiting under any circumstance. This corroborates the proposed predominance of the meizothrombin pathway when FXa is well saturated with the prothrombin complex. In contrast, the FXa-catalyzed hydrolysis of N-α-Z-D-Arg-Gly-Arg-pNA·2HCl (S-2765) and H-D-Ile-L-Pro-L-Arg-pNA.HCl (S-2288) is most consistent with two-proton bridges forming at the transition state between Ser195 OγH and His57 Nε2 and His57 Nδ1 and Asp102 COOβ- at the active site, with transition state fractionation factors of ϕ1 = ϕ2 = 0.57 ± 0.07 and ϕS = 0.78 ± 0.16 for solvent rearrangement for S-2765, and ϕ1 = ϕ2 = 0.674 ± 0.001 for S-2288 under enzyme saturation with substrate at pH 8.40 and 25.0 ± 0.1 °C. The rate-determining step(s) in these reactions is most likely the cleavage of the C-N bond and departure of the leaving group. PMID:17115712

  9. Acquired Factor V Inhibitor

    PubMed Central

    Hirai, Daisuke; Yamashita, Yugo; Masunaga, Nobutoyo; Katsura, Toshiaki; Akao, Masaharu; Okuno, Yoshiaki; Koyama, Hiroshi

    2016-01-01

    Inhibitors directed against factor V rarely occur, and the clinical symptoms vary. We herein report the case of a patient who presented with a decreased factor V activity that had decreased to <3 %. We administered vitamin K and 6 units of fresh frozen plasma, but she thereafter developed an intracerebral hemorrhage. It is unclear whether surgery >10 years earlier might have caused the development of a factor V inhibitor. The treatment of acquired factor V inhibitors is mainly the transfusion of platelet concentrates and corticosteroids. Both early detection and the early initiation of the treatment of factor V inhibitor are thus considered to be important. PMID:27746446

  10. Association Between Enoxaparin Dosage Adjusted by Anti-Factor Xa Trough Level and Clinically Evident Venous Thromboembolism After Trauma.

    PubMed

    Ko, Ara; Harada, Megan Y; Barmparas, Galinos; Chung, Kevin; Mason, Russell; Yim, Dorothy A; Dhillon, Navpreet; Margulies, Daniel R; Gewertz, Bruce L; Ley, Eric J

    2016-11-01

    Trauma patients are at high risk for developing venous thromboembolism (VTE). The VTE rate when enoxaparin sodium is dosed by anti-factor Xa (anti-Xa) trough level is not well described. To determine whether targeting a prophylactic anti-Xa trough level by adjusting the enoxaparin dose would reduce the VTE rate in trauma patients. Single-institution, historic vs prospective cohort comparison study at an urban, academic, level I trauma center. The prospective cohort was enrolled from August 2014 to May 2015 and compared with a historic cohort admitted from August 2013 to May 2014. Trauma patients who received enoxaparin adjusted by anti-Xa trough level (adjustment group) were compared with those who received enoxaparin sodium at a dosage of 30 mg twice daily (control group). Patients were excluded if they were younger than 18 years, had a length of hospital stay less than 2 days, or had preexisting deep vein thrombosis. Patients were excluded from the adjustment group for changes in the choice of thromboprophylaxis (heparin, enoxaparin once-daily dosing, early ambulation), hospital discharge before initial trough levels could be drawn, or incorrect timing of trough levels. Anti-Xa trough levels were monitored in patients in the adjustment group receiving 3 or more consecutive doses of enoxaparin sodium, 30 mg twice daily. Patients with a trough level of 0.1 IU/mL or lower received enoxaparin sodium increased by 10-mg increments. After providing 3 adjusted doses of enoxaparin, the trough level was redrawn and the dosage was adjusted as necessary. Patients in the control group received enoxaparin sodium at a dosage of 30 mg twice daily without adjustments. Rates of symptomatic VTE (deep vein thrombosis and pulmonary embolism, confirmed by duplex ultrasonography and chest computed tomographic angiography, respectively) and bleeding risk. A total of 205 patients (mean [SD] age, 41.3 [18.2] years; 75.1% male) were studied, 87 in the adjustment group and 118 in the

  11. Inhibitory properties of separate recombinant Kunitz-type-protease-inhibitor domains from tissue-factor-pathway inhibitor.

    PubMed

    Petersen, L C; Bjørn, S E; Olsen, O H; Nordfang, O; Norris, F; Norris, K

    1996-01-15

    Tissue-factor-pathway inhibitor (TFPI) is a multivalent inhibitor with three tandemly arranged Kunitz- type-protease-inhibitor (KPI) domains. Previous studies [Girard, Y. J., Warren, L. A., Novotny , W. F., Likert, K. M., Brown, S. G., Miletich, J. R & Broze, G. J. (1989) Nature 338, 518-520] by means of site-directed mutagenesis indicated that KPI domain 1 interacts with factor VIIa, that KPI domain 2 interacts with factor Xa, and that KPI domain 3 is apparently without inhibitory function. To elucidate the reaction mechanism of this complex inhibitor, we followed a different approach and studied the inhibitory properties of fragments of TFPI obtained by expression in yeast. Results obtained with TFPI-(1-161)-peptide and separate recombinant TFPI-KPI domains 1, 2 and 3 showed that KPI domain 1 inhibited factor VIIa/tissue factor (Ki = 250 nM), KPI domain 2 inhibited factor Xa (Ki = 90 nM), and that KPI domain 3 was without detectable inhibitory function. Studies with separate KPI domains also showed that KPI domain 2 was mainly responsible for inhibition of trypsin (Ki = 0.1 nM) and chymotrypsin (Ki = 0.75 nM), whereas KPI domain 1 inhibited plasmin (Ki = 26 nM) and cathepsin G (Ki = 200 nM). The structural basis for the interaction between serine proteases and KPI domains is discussed in terms of putative three-dimensional models of the proteins derived by comparative molecular-modelling methods. Studies of factor Xa inhibition by intact TFPI (Ki approximately 0.02 nM) suggested that regions other than the contact area of the KPI domain, interacted strongly with factor Xa. Secondary-site interactions were crucial for TFPI inhibition of factor Xa but was of little or no importance for its inhibition of trypsin.

  12. Differential Mitogenic Effects of Single Chain Hepatocyte Growth Factor (HGF)/Scatter Factor and HGF/NK1 following Cleavage by Factor Xa*

    PubMed Central

    Pediaditakis, Peter; Monga, Satdarshan P. S.; Mars, Wendy M.; Michalopoulos, George K.

    2007-01-01

    Hepatocyte growth factor/scatter factor (HGF/SF) is a multifunctional cytokine that is involved in many normal as well as pathological conditions. HGF/NK1, a splice variant of HGF/SF, has been reported to have either antagonistic or agonistic effects with regard to c-Met signaling depending on the cell type. In these experiments, we have determined that HGF/NK1 is a potent mitogen for rat hepatocytes in culture. Furthermore, we have found that coagulation factor Xa (fXa) is capable of cleaving HGF/NK1 and single chain HGF/SF (scHGF/SF). The products resulting from cleavage of HGF/NK1 or scHGF/SF by fXa appear as single bands under non-reducing conditions. The reaction products from the digestion of HGF/NK1 by fXa were separated under reducing conditions, and the cleavage site, as determined by N-terminal sequencing, was located C-terminal to arginine 134. Previous work established that the heparin-binding domain for HGF/SF is located in the N domain of HGF/SF. Additionally, the dimerization of the HGF/SF receptor (c-Met) by the ligand HGF/NK1 is facilitated by heparin and related sulfonated sugars on the cell surface, whereas heparin is not required for HGF/SF-mediated dimerization. Cleavage of single chain HGF/SF or HGF/NK1 by factor Xa does not alter the affinity of the respective molecules for heparin, but it did variably affect the associated mitogenic activity of these factors. The associated mitogenic activity of HGF/NK1 was reduced by more than 90%, whereas the mitogenic activity of scHGF/SF was unaffected. This suggests mandatory maintenance of a steric interaction of the N domain and the first kringle domain for HGF/NK1 to act as an agonist for rat hepatocyte growth but is not required by full-length HGF/SF. PMID:11832492

  13. Coagulation factor Xa drives tumor cells into apoptosis through BH3-only protein Bim up-regulation

    SciTech Connect

    Borensztajn, Keren S. . E-mail: K.S.Borensztajn@amc.uva.nl; Bijlsma, Maarten F.; Groot, Angelique P.; Brueggemann, Lois W.; Versteeg, Henri H.; Reitsma, Pieter H.; Peppelenbosch, Maikel P.; Spek, C. Arnold

    2007-07-15

    Coagulation Factor (F)Xa is a serine protease that plays a crucial role during blood coagulation by converting prothrombin into active thrombin. Recently, however, it emerged that besides this role in coagulation, FXa induces intracellular signaling leading to different cellular effects. Here, we show that coagulation factor (F)Xa drives tumor cells of epithelial origin, but not endothelial cells or monocytes, into apoptosis, whereas it even enhances fibroblast survival. FXa signals through the protease activated receptor (PAR)-1 to activate extracellular-signal regulated kinase (ERK) 1/2 and p38. This activation is associated with phosphorylation of the transcription factor CREB, and in tumor cells with up-regulation of the BH3-only pro-apoptotic protein Bim, leading to caspase-3 cleavage, the main hallmark of apoptosis. Transfection of tumor cells with dominant negative forms of CREB or siRNA for either PAR-1, Bim, ERK1 and/or p38 inhibited the pro-apoptotic effect of FXa. In fibroblasts, FXa-induced PAR-1 activation leads to down-regulation of Bim and pre-treatment with PAR-1 or Bim siRNA abolishes proliferation. We thus provide evidence that beyond its role in blood coagulation, FXa plays a key role in cellular processes in which Bim is the central player in determining cell survival.

  14. Andexanet alfa: a recombinant mimetic of human factor Xa for the reversal of anticoagulant therapies.

    PubMed

    Escolar, G; Diaz-Ricart, M; Arellano-Rodrigo, E

    2017-05-01

    Activated coagulation factor X (FXa) is a common target for classic and newer anticoagulants. Parenteral anticoagulants with an indirect inhibitory action on FXa (low-molecular-weight heparins) have a well-established clinical efficacy in the prophylaxis and therapy of thromboembolic conditions. More recently developed direct oral anticoagulants (DOACs) have emerged as a new class of antithrombotic drugs. Rivaroxaban, apixaban and edoxaban are direct inhibitors of FXa approved for the management of venous thromboembolism and stroke prevention in atrial fibrillation. Although these DOACs are associated with fewer hemorrhagic side effects than classic vitamin K antagonists, bleeding is still a main complication. FXa antagonists had no specific agents that could reverse their antihemostatic effects. Andexanet alfa is a modified, recombinant human FXa molecule with an enhanced ability to bind to both direct and indirect FXa inhibitors, but unable to contribute to blood coagulation mechanisms. Andexanet alfa is designed to reverse the anticoagulant effects of FXa inhibitors. This review will address the preclinical pharmacology and the main aspects of the clinical development of andexanet alfa for the reversal of anticoagulant therapies with an inhibitory action on FXa. It will also summarize additional completed or ongoing studies on andexanet alfa available to the scientific community until present. Copyright 2017 Clarivate Analytics.

  15. Factor Xa induces tissue factor expression in endothelial cells by P44/42 MAPK and NF-κB-dependent pathways

    PubMed Central

    Jiang, Rong; Wang, Ning-Ping; Tanaka, Kenichi A.; Levy, Jerrold H.; Guyton, Robert A.; Zhao, Zhi-Qing; Vinten-Johansen, Jakob

    2010-01-01

    Summary Background Tissue factor (TF) is an initiator of coagulation. The serine protease factor Xa (FXa) is the convergence point of the extrinsic and intrinsic components of the coagulation cascade. In addition to its hemostatic function, FXa elicits inflammatory responses in endothelial cells that may be important in surgical procedures in which inflammation is triggered. This study tested the hypothesis that FXa can upregulate TF on vascular endothelial cells by a MAPK- and NF-κB- dependent pathway. Methods and results Incubation of cultured human umbilical vein endothelial cells (HUVECs) with FXa increased TF protein expression and activity in a dose–dependent manner. Pre-incubation of HUVECs with the serine protease inhibitor antithrombin, which targets not only thrombin but also FXa and FIXa, inhibited FXa-induced TF expression, but the selective thrombin inhibitor hirudin did not inhibit FXa-induced TF expression, ruling out a thrombin-mediated pathway. After 10 min incubation with HUVECs, FXa rapidly induced P44/42 MAPK activation (immunoblotting of phosphorylated P44/42 MAPK) with a peak at 30 minutes. The MEK 1/2 inhibitor PD98059 partially reduced FXa-induced TF expression and activity (3.82±0.11 vs 6.54±0.08 fmol/min/cm2, P<0.05). NF-κB was activated by FXa, confirmed by cytoplasmic IkB α degradation and increased NF-κB P65 nuclear translocation. Interruption of the NF-κB pathway by the IkB α phosphorylation inhibitor Bay 11-7802 abrogated FXa-induced TF protein expression and activity (1.93± 0.02 vs 6.54±0.08 fmol/min/cm2, P<0.05). However, inhibition of PI3 kinase by LY 294002 did not attenuate FXa-induced TF protein expression and activity. Conclusions 1) FXa upregulates TF protein expression and activity in HUVECs 2) FXa-induced upregulation of TF is independent of the thrombin-PAR1 pathway, and 3) the MAPK and NF-kB pathways, but not PI3 kinase pathway, are involved in FXa-induced TF expression on human umbilical endothelial cells. FXa

  16. [Acquired coagulant factor inhibitors].

    PubMed

    Nogami, Keiji

    2015-02-01

    Acquired coagulation factor inhibitors are an autoimmune disease causing bleeding symptoms due to decreases in the corresponding factor (s) which result from the appearance of autoantibodies against coagulation factors (inhibitor). This disease is quite different from congenital coagulation factor deficiencies based on genetic abnormalities. In recent years, cases with this disease have been increasing, and most have anti-factor VIII autoantibodies. The breakdown of the immune control mechanism is speculated to cause this disease since it is common in the elderly, but the pathology and pathogenesis are presently unclear. We herein describe the pathology and pathogenesis of factor VIII and factor V inhibitors. Characterization of these inhibitors leads to further analysis of the coagulation process and the activation mechanisms of clotting factors. In the future, with the development of new clotting examination method (s), we anticipate that further novel findings will be obtained in this field through inhibitor analysis. In addition, detailed elucidation of the coagulation inhibitory mechanism possibly leading to hemostatic treatment strategies for acquired coagulation factor disorders will be developed.

  17. Daboxin P, a Major Phospholipase A2 Enzyme from the Indian Daboia russelii russelii Venom Targets Factor X and Factor Xa for Its Anticoagulant Activity

    PubMed Central

    Iyer, Janaki Krishnamurthy; Shih, Norrapat; Majumder, Munmi; Mattaparthi, Venkata Satish Kumar; Mukhopadhyay, Rupak; Doley, Robin

    2016-01-01

    In the present study a major protein has been purified from the venom of Indian Daboia russelii russelii using gel filtration, ion exchange and Rp-HPLC techniques. The purified protein, named daboxin P accounts for ~24% of the total protein of the crude venom and has a molecular mass of 13.597 kDa. It exhibits strong anticoagulant and phospholipase A2 activity but is devoid of any cytotoxic effect on the tested normal or cancerous cell lines. Its primary structure was deduced by N-terminal sequencing and chemical cleavage using Edman degradation and tandem mass spectrometry. It is composed of 121 amino acids with 14 cysteine residues and catalytically active His48 -Asp49 pair. The secondary structure of daboxin P constitutes 42.73% of α-helix and 12.36% of β-sheet. It is found to be stable at acidic (pH 3.0) and neutral pH (pH 7.0) and has a Tm value of 71.59 ± 0.46°C. Daboxin P exhibits anticoagulant effect under in-vitro and in-vivo conditions. It does not inhibit the catalytic activity of the serine proteases but inhibits the activation of factor X to factor Xa by the tenase complexes both in the presence and absence of phospholipids. It also inhibits the tenase complexes when active site residue (His48) was alkylated suggesting its non-enzymatic mode of anticoagulant activity. Moreover, it also inhibits prothrombinase complex when pre-incubated with factor Xa prior to factor Va addition. Fluorescence emission spectroscopy and affinity chromatography suggest the probable interaction of daboxin P with factor X and factor Xa. Molecular docking analysis reveals the interaction of the Ca+2 binding loop; helix C; anticoagulant region and C-terminal region of daboxin P with the heavy chain of factor Xa. This is the first report of a phospholipase A2 enzyme from Indian viper venom which targets both factor X and factor Xa for its anticoagulant activity. PMID:27089306

  18. Kinetics of the activation of human prothrombin by human coagulation factor Xa. Initial rate studies in the presence of Ca2+ and phospholipid.

    PubMed

    Kosow, D P; Orthner, C L

    1979-10-10

    Steady state kinetic studies have been performed to investigate the formation of thrombin from prothrombin by human coagulation Factor Xa in the presence of Ca2+ and phospholipid. The concentration of ligand which gives 50% of the maximum velocity (K0.5) is 2.3 mM for Ca2+, 7.4 microM for phospholipid, and 0.006 microM for prothrombin. Hill plots of the Ca2+ enhancement of the reaction give a Hill coefficient of 3.1, indicating positive cooperativity. The initial velocity patterns are consistent with an ordered addition of reactants with phospholipid as the second reactant to bind to the enzyme. Although our results do not differentiate between Ca2+ or the prothrombin substrate as the first reactant to bind to Factor Xa, it is established that Ca2+ can bind to Factor Xa in the absence of the other reactants. Thus, the most probable order of addition of reactants is Ca2+, phospholipid, and the prothrombin substrate. Plots of (v)-1 versus (prothrombin)-1 or (v)-1 versus [(Ca2+)3]-1 at several constant concentrations of phospholipid indicate that the major effect of phospholipid is to increase the turnover number of Factor Xa.

  19. Characterization of a human coagulation factor Xa-binding site on Viperidae snake venom phospholipases A2 by affinity binding studies and molecular bioinformatics

    PubMed Central

    Faure, Grazyna; Gowda, Veerabasappa T; Maroun, Rachid C

    2007-01-01

    Background The snake venom group IIA secreted phospholipases A2 (SVPLA2), present in the Viperidae snake family exhibit a wide range of toxic and pharmacological effects. They exert their different functions by catalyzing the hydrolysis of phospholipids (PL) at the membrane/water interface and by highly specific direct binding to: (i) presynaptic membrane-bound or intracellular receptors; (ii) natural PLA2-inhibitors from snake serum; and (iii) coagulation factors present in human blood. Results Using surface plasmon resonance (SPR) protein-protein interaction measurements and an in vitro biological test of inhibition of prothrombinase activity, we identify a number of Viperidae venom SVPLA2s that inhibit blood coagulation through direct binding to human blood coagulation factor Xa (FXa) via a non-catalytic, PL-independent mechanism. We classify the SVPLA2s in four groups, depending on the strength of their binding. Molecular electrostatic potentials calculated at the surface of 3D homology-modeling models show a correlation with inhibition of prothrombinase activity. In addition, molecular docking simulations between SVPLA2 and FXa guided by the experimental data identify the potential FXa binding site on the SVPLA2s. This site is composed of the following regions: helices A and B, the Ca2+ loop, the helix C-β-wing loop, and the C-terminal fragment. Some of the SVPLA2 binding site residues belong also to the interfacial binding site (IBS). The interface in FXa involves both, the light and heavy chains. Conclusion We have experimentally identified several strong FXa-binding SVPLA2s that disrupt the function of the coagulation cascade by interacting with FXa by the non-catalytic PL-independent mechanism. By theoretical methods we mapped the interaction sites on both, the SVPLA2s and FXa. Our findings may lead to the design of novel, non-competitive FXa inhibitors. PMID:18062812

  20. Assessment of anti-factor Xa activity of heparin in binary parenteral nutrition admixtures for premature neonates.

    PubMed

    Foinard, A; Perez, M; Barthélémy, C; Lannoy, D; Flamein, F; Storme, L; Tournoys, A; Décaudin, B; Odou, P

    2015-07-01

    An in vitro study was carried out to determine the anti-Xa activity of heparin in binary parenteral nutrition (BPN) admixtures for premature neonates in our neonatal intensive care unit (NICU) after a 24-hour infusion, as well as to assess drug interaction with a 50% glucose solution. Two types of bags were prepared: (1) BPN admixtures (composition defined in the NICU) including sodium heparin at 77 UI/mL and (2) bags containing only G50% with sodium heparin at 193 UI/mL. The anti-Xa activity of heparin was measured in bags at T0, after the 24-hour infusion and in eluates at the outlet of the infusion line after 24hours, using a validated chromogenic anti-Xa method. Comparisons of the mean concentration observed with the theoretical value for anti-Xa activity were performed with the Student t-test. Mean values of anti-Xa activity do not differ significantly from the values expected for all conditions. We found a slight variation in anti-Xa activity when infused over 24hours for both types of bags, with and without in-line filtration, showing that heparin remains stable during this infusion period in both BPN admixtures and G50%.

  1. Reproducibility of the anti-Factor Xa and anti-Factor IIa assays applied to enoxaparin solution.

    PubMed

    Martinez, Céline; Savadogo, Adama; Agut, Christophe; Anger, Pascal

    2013-01-01

    Enoxaparin is a widely used subcutaneously administered antithrombotic agent comprising a complex mixture of glycosaminoglycan chains. Owing to this complexity, its antithrombotic potency cannot be defined by physicochemical methods and is therefore evaluated using an enzymatic assay of anti-Xa and anti-IIa activity. Maintaining consistent anti-Xa activity in the final medicinal product allows physicians to ensure administration of the appropriate dosage to their patients. Bioassays are usually complex and display poorer reproducibility than physicochemical tests such as HPLC assays. Here, we describe the implementation of a common robotic platform and standard release potency testing procedures for enoxaparin sodium injection (Lovenox, Sanofi, Paris, France) products at seven quality control sites within Sanofi. Qualification and analytical procedures, as well as data handling, were optimized and harmonized to improve assay reproducibility. An inter-laboratory study was performed in routine-release conditions. The coefficients of variation for repeatability and reproducibility in assessments of anti-Xa activity were 1.0% and 1.2%, respectively. The tolerance interval in reproducibility precision conditions, expressed as percentage potency, was 96.8-103.2% of the drug product target of 10,000 IU/ml, comparing favorably with the United States of America Pharmacopeia specification (90-110%). The maximum difference between assays in two different laboratories is expected to be 4.1%. The reproducibility characteristics of anti-IIa activity assessments were found to be similar. These results demonstrate the effectiveness of the standardization process established and allow for further improvements to quality control in Lovenox manufacture. This process guarantees closeness between actual and target potencies, as exemplified by the results of release assays obtained during a three-year period.

  2. Global coagulation tests: their applicability for measuring direct factor Xa- and thrombin inhibition and reversal of anticoagulation by prothrombin complex concentrate.

    PubMed

    Dinkelaar, Jasper; Patiwael, Sanne; Harenberg, Job; Leyte, Anja; Brinkman, Herm Jan M

    2014-11-01

    Specific mass spectrometry and direct activated factor X (Xa)- and thrombin inhibition assays do not allow determination of the reversal of anticoagulant effects of non-vitamin K direct oral anticoagulants (NOACs) by prothrombin complex concentrate (PCC). The objective of this study was the evaluation of the applicability of a variety of commercially available global coagulation assays in analyzing the reversal of NOAC anticoagulation by PCC. Plasma and whole blood were spiked with apixaban or dabigatran and PCC was added to these samples. Prothrombin time (PT), modified PT (mPT), activated partial prothrombin time (APTT), thrombography (CAT method) and thromboelastography (ROTEM, TEG) were performed. Assays triggered by contact activation (APTT, INTEM) did not show inhibitor reversal by PCC. Assays triggered by tissue factor (TF) showed NOAC type and NOAC concentration dependent anticoagulation reversal effects of PCC ranging from partial normalization to overcorrection of the following parameters: clotting or reaction time (PT, mPT TEG-TF, EXTEM, FIBTEM); angle in thromboelastography (TEG-TF); thrombin generation (CAT) lag time, endogenous thrombin potential (ETP) and peak thrombin. Extent of reversal was assay reagent dependent. ETP (5 pM TF) was the only parameter showing complete reversal of anticoagulation by PCC for all NOACs ranging from 200 to 800 μg/L. ETP fits with the concept that reversal assessment of NOAC anticoagulation by PCC should be based on measurements on the clotting potential or thrombin generating potential of the plasma or whole blood patient sample. Low sensitivity of ETP for NOACs and its correlation with bleeding are issues that remain to be resolved.

  3. Independent anti-angiogenic capacities of coagulation factors X and Xa.

    PubMed

    Lange, Soledad; Gonzalez, Ibeth; Pinto, Mauricio P; Arce, Maximiliano; Valenzuela, Rodrigo; Aranda, Evelyn; Elliot, Matias; Alvarez, Marjorie; Henriquez, Soledad; Velasquez, Ethel V; Orge, Felipe; Oliva, Barbara; Gonzalez, Pamela; Villalon, Manuel; Cautivo, Kelly M; Kalergis, Alexis M; Pereira, Karla; Mendoza, Camila; Saez, Claudia; Kato, Sumie; Cuello, Mauricio A; Parborell, Fernanda; Irusta, Griselda; Palma, Veronica; Allende, Miguel L; Owen, Gareth I

    2014-11-01

    Knockout models have shown that the coagulation system has a role in vascular development and angiogenesis. Herein, we report for the first time that zymogen FX and its active form (FXa) possess anti-angiogenic properties. Both the recombinant FX and FXa inhibit angiogenesis in vitro using endothelial EA.hy926 and human umbilical cord vascular endothelial cells (HUVEC). This effect is dependent on the Gla domain of FX. We demonstrate that FX and FXa use different mechanisms: the use of Rivaroxaban (RX) a specific inhibitor of FXa attenuated its anti-angiogenic properties but did not modify the anti-angiogenic effect of FX. Furthermore, only the anti-angiogenic activity of FXa is PAR-1dependent. Using in vivo models, we show that FX and FXa are anti-angiogenic in the zebrafish intersegmental vasculature (ISV) formation and in the chick embryo chorioallantoic membrane (CAM) assays. Our results provide further evidence for the non-hemostatic functions of FX and FXa and demonstrate for the first time a biological role for the zymogen FX.

  4. Molecular recognition of avirulence protein (avrxa5) by eukaryotic transcription factor xa5 of rice (Oryza sativa L.): insights from molecular dynamics simulations.

    PubMed

    Dehury, Budheswar; Maharana, Jitendra; Sahoo, Bikash Ranjan; Sahu, Jagajjit; Sen, Priyabrata; Modi, Mahendra Kumar; Barooah, Madhumita

    2015-04-01

    The avirulence gene avrxa5 of bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) recognized by the resistant rice lines having corresponding resistance (xa5) gene in a gene-for-gene manner. We used a combinatorial approach involving protein-protein docking, molecular dynamics (MD) simulations and binding free energy calculations to gain novel insights into the gene-for-gene mechanism that governs the direct interaction of R-Avr protein. From the best three binding poses predicted by molecular docking, MD simulations were performed to explore the dynamic binding mechanism of xa5 and avrxa5. Molecular Mechanics/Poisson Boltzmann Surface Area (MM/PBSA) techniques were employed to calculate the binding free energy and to uncover the thriving force behind the molecular recognition of avrxa5 by eukaryotic transcription factor xa5. Binding free energy analysis revealed van der Waals term as the most constructive component that favors the xa5 and avrxa5 interaction. In addition, hydrogen bonds (H-bonds) and essential electrostatic interactions analysis highlighted amino acid residues Lys54/Asp870, Lys56/Ala868, Lys56/Ala866, Lys56/Glu871, Ile59/His862, Gly61/Phe858, His62/Arg841, His62/Leu856, Ser101/Ala872 and Ser105/Asp870 plays pivotal role for the energetically stability of the R-Avr complex. Insights gained from the present study are expected to unveil the molecular mechanisms that define the transcriptional activator mediated transcriptome modification in host plants. Copyright © 2015 Elsevier Inc. All rights reserved.

  5. Isolation and study of an acquired inhibitor of human coagulation factor V.

    PubMed Central

    Nesheim, M E; Nichols, W L; Cole, T L; Houston, J G; Schenk, R B; Mann, K G; Bowie, E J

    1986-01-01

    A coagulation Factor V inhibitor developed in a man 75 yr of age in association with an anaplastic malignancy and drug treatment (including the aminoglycoside antibiotic, gentamicin). The patient did not bleed abnormally, despite both surgical challenge and plasma Factor V activity of less than 1%. The inhibited plasma had grossly prolonged prothrombin and activated partial thromboplastin times, but a normal thrombin time. Mixing studies indicated progressive coagulation inhibition with normal plasma, but not with Factor V-deficient plasma, and reversal of coagulation inhibition by the addition of bovine Factor V to the patient's plasma. 1 ml of patient plasma inhibited the Factor V activity of 90 ml of normal human plasma. The inhibitor was isolated by sequential affinity chromatography on protein A-Sepharose and Factor V-Sepharose. The IgG isolate markedly inhibits the activity of prothrombinase assembled from purified Factors Xa and Va, calcium ion, and phospholipid vesicles, and partially inhibits prothrombinase assembled from purified Factor Xa, calcium ion, and normal platelets. The Factor V of platelets, however, appears relatively inaccessible to the antibody, inasmuch as platelets isolated from whole blood supplemented for 8 h with the antibody functioned normally with respect to platelet Factor V-mediated prothrombinase function. The absence of obvious hemorrhagic difficulties in the patient, the total inhibition of plasma Factor V by the inhibitor, and the apparent inaccessibility of platelet Factor V to the inhibitor specifically implicate platelet Factor V in the maintenance of hemostasis. Images PMID:3944265

  6. Novel thrombin and factor Xa inhibitors: challenges to reversal of their anticoagulation effects.

    PubMed

    Yates, Sean; Sarode, Ravi

    2013-11-01

    Warfarin has been the sole oral anticoagulant used in the management of thromboembolic disorders for over 60 years. Target-specific oral anticoagulants (TSOAs) have recently emerged as alternatives to warfarin, because they do not require laboratory monitoring. Nevertheless, with the rising use of TSOAs, there is growing concern among clinicians regarding management of bleeding in patients taking them. Unlike warfarin, there is no antidote or reversal agent for TSOAs. This review summarizes recent developments and attempts to provide a systematic approach to patients on TSOAs presenting with bleeding complications. Currently, data involving clinical management of TSOAs are limited and primarily based on ex-vivo or animal models using hemostatic agents with uncertain implications in bleeding patients. There is a pressing need for randomized clinical trials evaluating the safety and efficacy of hemostatic agents. Without evidence-based guidelines for TSOA management, appropriate patient care requires an understanding of TSOA pharmacology, their effect on coagulation tests and, hence, a correct interpretation of test results, as well as a systematic approach to bleeding complications.

  7. RoXaN, a Novel Cellular Protein Containing TPR, LD, and Zinc Finger Motifs, Forms a Ternary Complex with Eukaryotic Initiation Factor 4G and Rotavirus NSP3

    PubMed Central

    Vitour, Damien; Lindenbaum, Pierre; Vende, Patrice; Becker, Michelle M.; Poncet, Didier

    2004-01-01

    Rotavirus mRNAs are capped but not polyadenylated, and viral proteins are translated by the cellular translation machinery. This is accomplished through the action of the viral nonstructural protein NSP3, which specifically binds the 3′ consensus sequence of viral mRNAs and interacts with the eukaryotic translation initiation factor eIF4G I. To further our understanding of the role of NSP3 in rotavirus replication, we looked for other cellular proteins capable of interacting with this viral protein. Using the yeast two-hybrid assay, we identified a novel cellular protein-binding partner for rotavirus NSP3. This 110-kDa cellular protein, named RoXaN (rotavirus X protein associated with NSP3), contains a minimum of three regions predicted to be involved in protein-protein or nucleic acid-protein interactions. A tetratricopeptide repeat region, a protein-protein interaction domain most often found in multiprotein complexes, is present in the amino-terminal region. In the carboxy terminus, at least five zinc finger motifs are observed, further suggesting the capacity of RoXaN to bind other proteins or nucleic acids. Between these two regions exists a paxillin leucine-aspartate repeat (LD) motif which is involved in protein-protein interactions. RoXaN is capable of interacting with NSP3 in vivo and during rotavirus infection. Domains of interaction were mapped and correspond to the dimerization domain of NSP3 (amino acids 163 to 237) and the LD domain of RoXaN (amino acids 244 to 341). The interaction between NSP3 and RoXaN does not impair the interaction between NSP3 and eIF4G I, and a ternary complex made of NSP3, RoXaN, and eIF4G I can be detected in rotavirus-infected cells, implicating RoXaN in translation regulation. PMID:15047801

  8. Factor Xa releases matrix metalloproteinase-2 (MMP-2) from human vascular smooth muscle cells and stimulates the conversion of pro-MMP-2 to MMP-2: role of MMP-2 in factor Xa-induced DNA synthesis and matrix invasion.

    PubMed

    Rauch, Bernhard H; Bretschneider, Ellen; Braun, Marina; Schrör, Karsten

    2002-05-31

    Pro-matrix metalloproteinase-2 (pro-MMP-2) is expressed in vascular smooth muscle cells (SMCs). We report that activated coagulation factor X (FXa) induces the release of MMP-2 (65 kDa) from human SMCs. In addition, FXa cleaves pro-MMP-2 (72 kDa) into MMP-2. Pro-MMP-2 and MMP-2 were determined by gelatin zymography. MMP-2 was generated in conditioned medium containing pro-MMP-2 in a concentration-dependent fashion by FXa (3 to 100 nmol/L). FX at concentrations up to 300 nmol/L was ineffective. The conversion of pro-MMP-2 to MMP-2 was inhibited by a selective FXa inhibitor (DX-9065a) at 3 to 10 micromol/L. There was a concentration-dependent induction of an intermediate MMP-2 form (68 kDa) in lysates of FXa-treated cells. This indicates that cellular mechanisms are involved in FXa-induced conversion of pro-MMP-2. As a possible biological consequence of MMP-2 activation by FXa, DNA synthesis and matrix invasion of SMCs were determined. Both were stimulated by FXa and inhibited by the selective FXa inhibitor DX-9065a and the MMP inhibitor GM 6001 but not by hirudin or aprotinin. It is concluded that stimulation of SMCs by FXa increases the levels of MMP-2 in the extracellular space and that two different mechanisms are involved: release of active MMP-2 and cleavage of secreted pro-MMP-2. Both might contribute to the mitogenic potency of FXa and FXa-stimulated matrix invasion of SMCs.

  9. Staphylococcal superantigen-like protein 10 (SSL10) inhibits blood coagulation by binding to prothrombin and factor Xa via their γ-carboxyglutamic acid (Gla) domain.

    PubMed

    Itoh, Saotomo; Yokoyama, Ryosuke; Kamoshida, Go; Fujiwara, Toshinobu; Okada, Hiromi; Takii, Takemasa; Tsuji, Tsutomu; Fujii, Satoshi; Hashizume, Hideki; Onozaki, Kikuo

    2013-07-26

    The staphylococcal superantigen-like protein (SSL) family is composed of 14 exoproteins sharing structural similarity with superantigens but no superantigenic activity. Target proteins of four SSLs have been identified to be involved in host immune responses. However, the counterparts of other SSLs have been functionally uncharacterized. In this study, we have identified porcine plasma prothrombin as SSL10-binding protein by affinity purification using SSL10-conjugated Sepharose. The resin recovered the prodomain of prothrombin (fragment 1 + 2) as well as factor Xa in pull-down analysis. The equilibrium dissociation constant between SSL10 and prothrombin was 1.36 × 10(-7) M in surface plasmon resonance analysis. On the other hand, the resin failed to recover γ-carboxyglutamic acid (Gla) domain-less coagulation factors and prothrombin from warfarin-treated mice, suggesting that the Gla domain of the coagulation factors is essential for the interaction. SSL10 prolonged plasma clotting induced by the addition of Ca(2+) and factor Xa. SSL10 did not affect the protease activity of thrombin but inhibited the generation of thrombin activity in recalcified plasma. S. aureus produces coagulase that non-enzymatically activates prothrombin. SSL10 attenuated clotting induced by coagulase, but the inhibitory effect was weaker than that on physiological clotting, and SSL10 did not inhibit protease activity of staphylothrombin, the complex of prothrombin with coagulase. These results indicate that SSL10 inhibits blood coagulation by interfering with activation of coagulation cascade via binding to the Gla domain of coagulation factor but not by directly inhibiting thrombin activity. This is the first finding that the bacterial protein inhibits blood coagulation via targeting the Gla domain of coagulation factors.

  10. Proteinase inhibitors from the medicinal leech Hirudo medicinalis.

    PubMed

    Baskova, I P; Zavalova, L L

    2001-07-01

    The medicinal leech Hirudo medicinalis produces various types of proteinase inhibitors: bdellins (inhibitors of trypsin, plasmin, and acrosin), hirustasin (inhibitor of tissue kallikrein, trypsin, alpha-chymotrypsin, and granulocyte cathepsin G), tryptase inhibitor, eglins (inhibitors of alpha-chymotrypsin, subtilisin, and chymasin and the granulocyte proteinases elastase and cathepsin G), inhibitor of factor Xa, hirudin (thrombin inhibitor), inhibitor of carboxypeptidase, and inhibitor of complement component C1s. This review summarizes data on their primary and tertiary structures, action mechanisms, and biological activities.

  11. Homology modeling and structural validation of tissue factor pathway inhibitor

    PubMed Central

    Agrawal, Piyush; Thakur, Zoozeal; Kulharia, Mahesh

    2013-01-01

    Blood coagulation is a cascade of complex enzymatic reactions which involves specific proteins and cellular components to interact and prevent blood loss. The coagulation process begins by either “Tissue Dependent Pathway” (also known as extrinsic pathway) or by “contact activation pathway” (also known as intrinsic pathway). TFPI is an endogenous multivalent Kunitz type protease inhibitor which inhibits Tissue factor dependent pathway by inhibiting Tissue Factor:Factor VIIa (TF:FVIIa) complex and Factor Xa. TFPI is one of the most studied coagulation pathway inhibitor which has various clinical and potential therapeutic applications, however, its exact mechanism of inhibition is still unknown. Structure based mechanism elucidation is commonly employed technique in such cases. Therefore, in the current study the generated a complete TFPI structural model so as to understand the mechanistic details of it's functioning. The model was checked for stereochemical quality by PROCHECK-NMR, WHATIF, ProSA, and QMEAN servers. The model was selected, energy minimized and simulated for 1.5ns. The result of the study may be a guiding point for further investigations on TFPI and its role in coagulation mechanism. PMID:24143050

  12. Monitoring Low Molecular Weight Heparins at Therapeutic Levels: Dose-Responses of, and Correlations and Differences between aPTT, Anti-Factor Xa and Thrombin Generation Assays

    PubMed Central

    Thomas, Owain; Lybeck, Emanuel; Strandberg, Karin; Tynngård, Nahreen; Schött, Ulf

    2015-01-01

    Background Low molecular weight heparins (LMWH’s) are used to prevent and treat thrombosis. Tests for monitoring LMWH’s include anti-factor Xa (anti-FXa), activated partial thromboplastin time (aPTT) and thrombin generation. Anti-FXa is the current gold standard despite LMWH’s varying affinities for FXa and thrombin. Aim To examine the effects of two different LMWH’s on the results of 4 different aPTT-tests, anti-FXa activity and thrombin generation and to assess the tests’ concordance. Method Enoxaparin and tinzaparin were added ex-vivo in concentrations of 0.0, 0.5, 1.0 and 1.5 anti-FXa international units (IU)/mL, to blood from 10 volunteers. aPTT was measured using two whole blood methods (Free oscillation rheometry (FOR) and Hemochron Jr (HCJ)) and an optical plasma method using two different reagents (ActinFSL and PTT-Automat). Anti-FXa activity was quantified using a chromogenic assay. Thrombin generation (Endogenous Thrombin Potential, ETP) was measured on a Ceveron Alpha instrument using the TGA RB and more tissue-factor rich TGA RC reagents. Results Methods’ mean aPTT at 1.0 IU/mL LMWH varied between 54s (SD 11) and 69s (SD 14) for enoxaparin and between 101s (SD 21) and 140s (SD 28) for tinzaparin. ActinFSL gave significantly shorter aPTT results. aPTT and anti-FXa generally correlated well. ETP as measured with the TGA RC reagent but not the TGA RB reagent showed an inverse exponential relationship to the concentration of LMWH. The HCJ-aPTT results had the weakest correlation to anti-FXa and thrombin generation (Rs0.62–0.87), whereas the other aPTT methods had similar correlation coefficients (Rs0.80–0.92). Conclusions aPTT displays a linear dose-respone to LMWH. There is variation between aPTT assays. Tinzaparin increases aPTT and decreases thrombin generation more than enoxaparin at any given level of anti-FXa activity, casting doubt on anti-FXa’s present gold standard status. Thrombin generation with tissue factor-rich activator is

  13. The Levels of Tissue Factor Pathway Inhibitor in Sepsis Patients Receiving Prophylactic Enoxaparin

    PubMed Central

    Al Otair, Hadil A.; Abdel Gader, Abdel Galil M.; Khurshid, Syed M.; Alzeer, Abdulaziz H.; Al Momen, Abdul Kareem; Al Shaikh, Mashael; Al Gahtani, Farja; Al Aseri, Zohair A.; Abdelrazik, Hossam A.H.

    2016-01-01

    Objective: Sepsis syndrome is usually accompanied by activation of blood coagulation mechanisms. Earlier studies found deficiencies of the 3 main natural anticoagulants, antithrombin, protein C, and protein S. However, none of these inhibitors block tissue factor, the prime trigger of coagulation during sepsis that is controlled specifically by the tissue factor pathway inhibitor (TFPI). The aim of this study was to characterize the fluctuations in the levels of natural anticoagulants, particularly TFPI, in the course of sepsis and to find out their association with the anticoagulant action of the low-molecular-weight heparin enoxaparin. Materials and Methods: We studied 51 consecutive patients with sepsis. Blood samples were collected from patients at baseline (0 h) and at 4, 12, and 24 h after enoxaparin administration. The following assays were undertaken using commercial kits: activated partial thromboplastin time, prothrombin time, thrombin time, total and free TFPI, protein C and protein S, antithrombin, fibrinogen, and anti-factor Xa. Results: Before enoxaparin administration, there was significant prolongation of the prothrombin time and activated partial thromboplastin time, and this remained the case in the 3 subsequent samples. There was marked reduction in the levels of antithrombin, protein C, and total and free protein S to below control values throughout the study. In contrast, plasma levels of both total and free TFPI were markedly elevated and increased after enoxaparin therapy. Anti-factor Xa levels were within the therapeutic range throughout. There was no difference in TFPI levels between those patients who died and those who survived. Conclusion: Sepsis triggered marked release of TFPI from endothelial cells. This persisted and was increased further following the administration of enoxaparin. In contrast, there was marked consumption of the natural coagulation inhibitors antithrombin, protein C, and protein S. These results go some way towards

  14. Factor Xa activation of factor V is of paramount importance in initiating the coagulation system: lessons from a tick salivary protein.

    PubMed

    Schuijt, Tim J; Bakhtiari, Kamran; Daffre, Sirlei; Deponte, Kathleen; Wielders, Simone J H; Marquart, J Arnoud; Hovius, Joppe W; van der Poll, Tom; Fikrig, Erol; Bunce, Matthew W; Camire, Rodney M; Nicolaes, Gerry A F; Meijers, Joost C M; van 't Veer, Cornelis

    2013-07-16

    Generation of active procoagulant cofactor factor Va (FVa) and its subsequent association with the enzyme activated factor X (FXa) to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation, and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied with the use of plasma, whole blood, and purified systems. Here, we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. Accordingly, tick feeding is impaired on TIX-5 immune rabbits, displaying the in vivo importance of TIX-5. Our data elucidate a unique molecular mechanism by which ticks inhibit the host's coagulation system. From our data, we propose a revised blood coagulation scheme in which direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors.

  15. Regulation of factor IXa in vitro in human and mouse plasma and in vivo in the mouse. Role of the endothelium and the plasma proteinase inhibitors

    SciTech Connect

    Fuchs, H.E.; Trapp, H.G.; Griffith, M.J.; Roberts, H.R.; Pizzo, S.V.

    1984-06-01

    The regulation of human Factor IXa was studied in vitro in human and mouse plasma and in vivo in the mouse. In human plasma, approximately 60% of the /sup 125/I-Factor IXa was bound to antithrombin III (ATIII) by 2 h, with no binding to alpha 2-macroglobulin or alpha 1-proteinase inhibitor, as assessed by gel electrophoresis and IgG- antiproteinase inhibitor-Sepharose beads. In the presence of heparin, virtually 100% of the /sup 125/I-Factor IXa was bound to ATIII by 1 min. The distribution of /sup 125/I-Factor IXa in mouse plasma was similar. The clearance of /sup 125/I-Factor IXa was rapid (50% clearance in 2 min) and biphasic and was inhibited by large molar excesses of ATIII-thrombin and alpha 1-proteinase inhibitor-trypsin, but not alpha 2-macro-globulin-trypsin; it was also inhibited by large molar excesses of diisopropylphosphoryl - (DIP-) Factor Xa, DIP-thrombin, and Factor IX, but not by prothrombin or Factor X. The clearance of Factor IX was also rapid (50% clearance in 2.5 min) and was inhibited by a large molar excess of Factor IX, but not by large molar excesses of Factor X, prothrombin, DIP-Factor Xa, or DIP-thrombin. Electrophoresis and IgG- antiproteinase inhibitor-Sepharose bead studies confirmed that by 2 min after injection into the murine circulation, 60% of the /sup 125/I-Factor IXa was bound to ATIII. Organ distribution studies with /sup 125/I-Factor IXa demonstrated that most of the radioactivity was in the liver. These studies suggest that Factor IXa binds to at least two classes of binding sites on endothelial cells. One site apparently recognizes both Factors IX and IXa, but not Factor X, Factor Xa, prothrombin, or thrombin. The other site recognizes thrombin, Factor Xa, and Factor IXa, but not the zymogen forms of these clotting factors. After this binding, Factor IXa is bound to ATIII and the complex is cleared from the circulation by hepatocytes.

  16. Discovery of novel heterocyclic factor VIIa inhibitors.

    PubMed

    Rai, Roopa; Kolesnikov, Aleksandr; Sprengeler, Paul A; Torkelson, Steven; Ton, Tony; Katz, Bradley A; Yu, Christine; Hendrix, John; Shrader, William D; Stephens, Robin; Cabuslay, Ronnell; Sanford, Ellen; Young, Wendy B

    2006-04-15

    Structure-activity relationships and binding mode of novel heterocyclic factor VIIa inhibitors will be described. In these inhibitors, a highly basic 5-amidinoindole moiety has been successfully replaced with a less basic 5-aminopyrrolo[3,2-b]pyridine scaffold.

  17. Improvement of low bioavailability of a novel factor Xa inhibitor through formulation of cationic additives in its oral dosage form.

    PubMed

    Fujii, Yoshimine; Kanamaru, Taro; Kikuchi, Hiroshi; Nakagami, Hiroaki; Yamashita, Shinji; Akashi, Mitsuru; Sakuma, Shinji

    2011-12-15

    A clinical trial of (2S)-2-[4-[[(3S)-1-acetimidoyl-3-pyrrolidinyl]oxy]phenyl]-3-(7-amidino-2-naphtyl) propanoic acid (DX-9065) revealed that its oral bioavailability was only 3% when it was administered as a conventional capsule formulation. The low bioavailability of DX-9065 was likely caused by both its poor membrane permeability and its electrostatic interaction with anionic bile acids. We hypothesized that DX-9065 absorption would be enhanced when the cationic drug was free from the complex through its replacement with other cationic substances. Polystyrene nanospheres coated with cationic poly(vinylamine) and cholestyramine, which is clinically used as a cholesterol-lowering agent, dramatically prevented DX-9065 from interacting with chenodeoxycholic acid in vitro. Successive animal experiments showed that bioavailability of DX-9065 administered with these cationic substances was 2-3 times that of DX-9065 administered solely. A dry syrup formulation with one-half of a minimal cholesterol-lowering equivalent dose of cholestyramine was designed, and the clinical trial was resumed. A 1.3-fold increase in bioavailability of DX-9065 was observed when the dry syrup was administered. We successfully demonstrated that DX-9065 absorption was enhanced when the drug was administered with cationic additives; however, it appeared that the absorption-enhancing function of cholestyramine largely depended on its dose. The dose escalation is probably prerequisite for the significant improvement of DX-9065 absorption in humans.

  18. Comparison of postoperative complications after total hip arthroplasty among patients receiving aspirin, enoxaparin, warfarin, and factor Xa inhibitors.

    PubMed

    Agaba, Perez; Kildow, Beau J; Dhotar, Herman; Seyler, Thorsten M; Bolognesi, Michael

    2017-12-01

    Optimal prophylaxis for prevention of venous thromboembolism (VTE) after total joint arthroplasty (TJA) remains debated. The purpose of this study was to compare postoperative complications in patients receiving different VTE chemoprophylactic regimens. Using a nationwide healthcare database, 72,670 THA patients without a history of VTE were identified. Study cohorts received VTE prophylaxis within 30 days postoperatively. Odds ratios and 95% confidence intervals were used to assess 30-day and 90-day postoperative complications (hematoma, hemorrhage, transfusion, pulmonary embolism (PE), VTE, prosthetic joint infection (PJI), and incision/drainage (I&D)). Of the 72,670 THA patients, 25,966 received single medication VTE prophylaxis; 551 (2.12%) aspirin, 6791 (26.15%) enoxaparin, 12,008 (46.25%) warfarin, 5403 (20.81%) rivaroxaban, 876 (3.37%) fondaparinux and 337 (1.30%) apixaban. 30-day complications included; aspirin: I&D; warfarin: I&D, hematoma, hemorrhage, transfusion, PJI, PE and DVT; apixaban: hematoma and hemorrhage. 90-day complications included; aspirin: I&D; warfarin: I&D, hematoma, hemorrhage, transfusion, PJI, PE and DVT. Warfarin was the only anticoagulant associated with a higher risk for DVT, and the highest risk for 30-day and 90-day complications. Aspirin had the highest risk for I&D. Despite three times increased 30-day risk for bleeding, apixaban was effective in preventing VTE during the high-risk 3-month-period. Enoxaparin had the lowest risk for PE and DVT while rivaroxaban had the lowest risk for PJI, hematoma, I&D, hemorrhage and transfusion.

  19. Association between edoxaban dose, concentration, anti-Factor Xa activity, and outcomes: an analysis of data from the randomised, double-blind ENGAGE AF-TIMI 48 trial.

    PubMed

    Ruff, Christian T; Giugliano, Robert P; Braunwald, Eugene; Morrow, David A; Murphy, Sabina A; Kuder, Julia F; Deenadayalu, Naveen; Jarolim, Petr; Betcher, Joshua; Shi, Minggao; Brown, Karen; Patel, Indravadan; Mercuri, Michele; Antman, Elliott M

    2015-06-06

    New oral anticoagulants for stroke prevention in atrial fibrillation were developed to be given in fixed doses without the need for the routine monitoring that has hindered usage and acceptance of vitamin K antagonists. A concern has emerged, however, that measurement of drug concentration or anticoagulant activity might be needed to prevent excess drug concentrations, which significantly increase bleeding risk. In the ENGAGE AF-TIMI 48 trial, higher-dose and lower-dose edoxaban were compared with warfarin in patients with atrial fibrillation. Each regimen incorporated a 50% dose reduction in patients with clinical features known to increase edoxaban drug exposure. We aim to assess whether adjustment of edoxaban dose in this trial prevented excess drug concentration and the risk of bleeding events. We analysed data from the randomised, double-blind ENGAGE AF-TIMI 48 trial. We correlated edoxaban dose, plasma concentration, and anti-Factor Xa (FXa) activity and compared efficacy and safety outcomes with warfarin stratified by dose reduction status. Patients with atrial fibrillation and at moderate to high risk of stroke were randomly assigned in a 1:1:1 ratio to receive warfarin, dose adjusted to an international normalised ratio of 2·0-3·0, higher-dose edoxaban (60 mg once daily), or lower-dose edoxaban (30 mg once daily). Randomisation was done with use of a central, 24 h, interactive, computerised response system. International normalised ratio was measured using an encrypted point-of-care device. To maintain masking, sham international normalised ratio values were generated for patients assigned to edoxaban. Edoxaban (or placebo-edoxaban in warfarin group) doses were halved at randomisation or during the trial if patients had creatinine clearance 30-50 mL/min, bodyweight 60 kg or less, or concomitant medication with potent P-glycoprotein interaction. Efficacy outcomes included the primary endpoint of all-cause stroke or systemic embolism, ischaemic stroke, and

  20. Bacithrocins A, B and C, novel thrombin inhibitors.

    PubMed

    Kamiyama, T; Umino, T; Nakamura, Y; Itezono, Y; Sawairi, S; Satoh, T; Yokose, K

    1994-09-01

    Novel thrombin inhibitors, bacithrocins A, B and C, have been isolated from the culture broth of Bacillus laterosporus Laubach NR 2988. The structures of these inhibitors have been determined to be N-acyl-L-phenylalanyl-DL-arginal by the 2D-NMR experiments on their oxidation products and by amino acid analysis. Bacithrocin A inhibits thrombin, factor Xa and trypsin with IC50s of 48, 13 and 0.65 microM, respectively, which are similar to those of bacithrocins B and C. Bacithrocins prolong the clotting time induced by thrombin and factor Xa.

  1. Activated partial thromboplastin time and anti-xa measurements in heparin monitoring: biochemical basis for discordance.

    PubMed

    Takemoto, Clifford M; Streiff, Michael B; Shermock, Kenneth M; Kraus, Peggy S; Chen, Junnan; Jani, Jayesh; Kickler, Thomas

    2013-04-01

    We examined the concordance of heparin levels measured by a chromogenic anti-Xa assay and the activated partial thromboplastin time (APTT) during unfractionated heparin therapy (UFH) and the biochemical basis for differences between these measures. We also investigated the endogenous thrombin potential (ETP) as a possible measure of anticoagulation. Paired measures of anti-Xa and APTT were performed on 569 samples from 149 patients on UFH. The anti-Xa values and the APTT were concordant in only 54% of measurements. One hundred twelve samples from 59 patients on UFH were assayed for APTT, anti-Xa, factor II, factor VIII, and ETP. Supratherapeutic APTT values but therapeutic anti-Xa results had decreased factor II activity. Subtherapeutic APTT but therapeutic anti-Xa values had high factor VIII activity. ETP correlated with anticoagulation status and UFH dose. In conclusion, factor II and VIII activity contributes to discordance between APTT and anti-Xa results. ETP measurements may provide an additional assessment of anticoagulation status.

  2. Factor Xa Activation of Factor V is of Paramount Importance in Initiating the Coagulation System: Lessons from a Tick Salivary Protein

    PubMed Central

    Schuijt, Tim J.; Bakhtiari, Kamran; Daffre, Sirlei; DePonte, Kathleen; Wielders, Simone J.H.; Marquart, J. Arnoud; Hovius, Joppe W.; van der Poll, Tom; Fikrig, Erol; Bunce, Matthew W.; Camire, Rodney M.; Nicolaes, Gerry A.F.; Meijers, Joost C.M.; van 't Veer, Cornelis

    2013-01-01

    Background Generation of active procoagulant cofactor FVa and its subsequent association with the enzyme FXa to form the prothrombinase complex is a pivotal initial event in blood coagulation and has been the subject of investigative effort, speculation and controversy. The current paradigm assumes that FV activation is initiated by limited proteolysis by traces of (meizo) thrombin. Methods and Results Recombinant tick salivary protein TIX-5 was produced and anticoagulant properties were studied using plasma, whole blood and purified systems. Here we report that TIX-5 specifically inhibits FXa-mediated FV activation involving the B-domain of FV and show that FXa activation of FV is pivotal for plasma and blood clotting. In line, tick feeding is impaired on TIX-5 immune rabbits displaying the in vivo importance of TIX-5. Conclusions Our data elucidate a unique molecular mechanism by which ticks inhibit the host's coagulation system. Based on our data we propose a revised blood coagulation scheme wherein direct FXa-mediated FV activation occurs in the initiation phase during which thrombin-mediated FV activation is restrained by fibrinogen and inhibitors. PMID:23817575

  3. Inhibitors of the Metalloproteinase Anthrax Lethal Factor

    PubMed Central

    Goldberg, Allison B.; Turk, Benjamin E.

    2016-01-01

    Bacillus anthracis, a rod shaped, spore forming, gram positive bacteria, is the etiological agent of anthrax. B. anthracis virulence is partly attributable to two secreted bipartite protein toxins, which act inside host cells to disrupt signaling pathways important for host defense against infection. These toxins may also directly contribute to mortality in late stage infection. The zinc-dependent metalloproteinase anthrax lethal factor (LF) is a critical component of one of these protein toxins and a prime target for inhibitor development to produce anthrax therapeutics. Here, we describe recent efforts to identify specific and potent LF inhibitors. Derivatization of peptide substrate analogs bearing zinc-binding groups has produced potent and specific LF inhibitors, and X-ray crystallography of LF-inhibitor complexes has provided insight into features required for high affinity binding. Novel inhibitor scaffolds have been identified through several approaches, including fragment-based drug discovery, virtual screening, and high-throughput screening of diverse compound libraries. Lastly, efforts to discover LF inhibitors have led to the development of new screening strategies, such as the use of full-length proteins as substrates, that may prove useful for other proteases as well. Overall, these efforts have led to a collection of chemically and mechanistically diverse molecules capable of inhibiting LF activity in vitro and in cells, as well as in animal models of anthrax infection. PMID:27072692

  4. Structural mechanism for heparin-binding of the third Kunitz domain of human tissue factor pathway inhibitor.

    PubMed

    Mine, Shouhei; Yamazaki, Toshio; Miyata, Toshiyuki; Hara, Saburo; Kato, Hisao

    2002-01-08

    Tissue factor pathway inhibitor (TFPI) inhibits the activity of coagulation factor VIIa and Xa through its K1 and K2 domain, respectively, and the inhibitory activity is enhanced by heparin. The function of the K3 domain of TFPI has not been established, but the domain probably harbors a heparin binding site (HBS-2). We determined the three-dimensional solution structure of the TFPI K3 domain (Glu182-Gly242) by heteronuclear multidimensional NMR. The results showed that the molecule is composed of one antiparallel beta-sheet and one alpha-helix, and in overall structure is very similar to the K2 domain, with the rms deviation of 1.55 A for the 58 defined C(alpha) positions. However, the surface electrostatic properties of both domains are different each other. The lack of inhibitory activity of the K3 domain is explained by the absence of electrostatic interaction with factor Xa over a large surface area. A titration experiment with size-fractionated heparin showed that a heparin binding site was located in the vicinity of the alpha-helix. In this region, a positively charged cluster is formed by Lys213, Lys232, and Lys240, and the negatively charged sulfate groups of heparin bind there. The enhancement of inhibitory activity by heparin probably was not due to a conformational change to TFPI itself. It is likely that heparin simply increases the local concentration of TFPI on the cell surface and stabilizes the initial complex that forms.

  5. Designing Allosteric Inhibitors of Factor XIa. Lessons from the Interactions of Sulfated Pentagalloylglucopyranosides

    PubMed Central

    2015-01-01

    We recently introduced sulfated pentagalloylglucopyranoside (SPGG) as an allosteric inhibitor of factor XIa (FXIa) (Al-Horani et al., J. Med Chem.2013, 56, 867–87823316863). To better understand the SPGG–FXIa interaction, we utilized eight SPGG variants and a range of biochemical techniques. The results reveal that SPGG’s sulfation level moderately affected FXIa inhibition potency and selectivity over thrombin and factor Xa. Variation in the anomeric configuration did not affect potency. Interestingly, zymogen factor XI bound SPGG with high affinity, suggesting its possible use as an antidote. Acrylamide quenching experiments suggested that SPGG induced significant conformational changes in the active site of FXIa. Inhibition studies in the presence of heparin showed marginal competition with highly sulfated SPGG variants but robust competition with less sulfated variants. Resolution of energetic contributions revealed that nonionic forces contribute nearly 87% of binding energy suggesting a strong possibility of specific interaction. Overall, the results indicate that SPGG may recognize more than one anion-binding, allosteric site on FXIa. An SPGG molecule containing approximately 10 sulfate groups on positions 2 through 6 of the pentagalloylglucopyranosyl scaffold may be the optimal FXIa inhibitor for further preclinical studies. PMID:24844380

  6. Inhibitors of the Metalloproteinase Anthrax Lethal Factor.

    PubMed

    Goldberg, Allison B; Turk, Benjamin E

    2016-01-01

    Bacillus anthracis, a rod shaped, spore forming, gram positive bacteria, is the etiological agent of anthrax. B. anthracis virulence is partly attributable to two secreted bipartite protein toxins, which act inside host cells to disrupt signaling pathways important for host defense against infection. These toxins may also directly contribute to mortality in late stage infection. The zinc-dependent metalloproteinase anthrax lethal factor (LF) is a critical component of one of these protein toxins and a prime target for inhibitor development to produce anthrax therapeutics. Here, we describe recent efforts to identify specific and potent LF inhibitors. Derivatization of peptide substrate analogs bearing zinc-binding groups has produced potent and specific LF inhibitors, and X-ray crystallography of LFinhibitor complexes has provided insight into features required for high affinity binding. Novel inhibitor scaffolds have been identified through several approaches, including fragment-based drug discovery, virtual screening, and highthroughput screening of diverse compound libraries. Lastly, efforts to discover LF inhibitors have led to the development of new screening strategies, such as the use of full-length proteins as substrates, that may prove useful for other proteases as well. Overall, these efforts have led to a collection of chemically and mechanistically diverse molecules capable of inhibiting LF activity in vitro and in cells, as well as in animal models of anthrax infection.

  7. Monitoring of anti-Xa activity and factors related to bleeding events: A study in Japanese patients with nonvalvular atrial fibrillation receiving rivaroxaban.

    PubMed

    Sakaguchi, Teruhiro; Osanai, Hiroyuki; Murase, Yosuke; Ishii, Hideki; Nakashima, Yoshihito; Asano, Hiroshi; Suzuki, Susumu; Takefuji, Mikito; Inden, Yasuya; Sakai, Kazuyoshi; Murohara, Toyoaki; Ajioka, Masayoshi

    2017-09-01

    Anti-Xa activity (AXA) in patients with nonvalvular atrial fibrillation (NVAF) and relationship to bleeding events remains unclear. We evaluated AXA in 94 patients at both trough and peak rivaroxaban concentrations. Rivaroxaban dosage was determined according to creatinine clearance (CrCl): 10 and 15mg once daily for patients with CrCl 15-49 and CrCl ≥50mL/min, respectively. AXA value distribution and its association with bleeding events were examined in enrolled subjects. The mean peak AXA level was significantly higher than the mean trough level (1.98±0.81 vs. 0.16±0.15IU/mL; p<0.001). The peak AXA level significantly differed among patients with CrCl 15-29, 30-49, 50-79, and ≥80mL/min (2.51±0.83, 1.72±0.76, 2.05±0.82, and 1.66±0.51IU/mL, respectively; p=0.004). Major and non-major clinically relevant bleeding events occurred in 22 patients (23.4% and 14.6% per year, respectively). The mean peak AXA level was significantly higher in patients who experienced bleeding events than in those who did not (2.40±0.70 vs. 1.84±0.80IU/mL; p=0.001). A Cox multivariate analysis showed that the peak AXA level was independently related to the incidence of major and non-major clinically relevant bleeding events (p=0.012). Cumulative bleeding rates were significantly higher in patients with high peak AXA levels (p<0.001). Peak AXA level was an independent predictor for bleeding events in Japanese NVAF patients receiving rivaroxaban. Copyright © 2016 Japanese College of Cardiology. Published by Elsevier Ltd. All rights reserved.

  8. Efficacy and safety of rivaroxaban compared with warfarin among elderly patients with nonvalvular atrial fibrillation in the Rivaroxaban Once Daily, Oral, Direct Factor Xa Inhibition Compared With Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF).

    PubMed

    Halperin, Jonathan L; Hankey, Graeme J; Wojdyla, Daniel M; Piccini, Jonathan P; Lokhnygina, Yuliya; Patel, Manesh R; Breithardt, Günter; Singer, Daniel E; Becker, Richard C; Hacke, Werner; Paolini, John F; Nessel, Christopher C; Mahaffey, Kenneth W; Califf, Robert M; Fox, Keith A A

    2014-07-08

    Nonvalvular atrial fibrillation is common in elderly patients, who face an elevated risk of stroke but difficulty sustaining warfarin treatment. The oral factor Xa inhibitor rivaroxaban was noninferior to warfarin in the Rivaroxaban Once Daily, Oral, Direct Factor Xa Inhibition Compared With Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF). This prespecified secondary analysis compares outcomes in older and younger patients. There were 6229 patients (44%) aged ≥75 years with atrial fibrillation and ≥2 stroke risk factors randomized to warfarin (target international normalized ratio=2.0-3.0) or rivaroxaban (20 mg daily; 15 mg if creatinine clearance <50 mL/min), double blind. The primary end point was stroke and systemic embolism by intention to treat. Over 10 866 patient-years, older participants had more primary events (2.57% versus 2.05%/100 patient-years; P=0.0068) and major bleeding (4.63% versus 2.74%/100 patient-years; P<0.0001). Stroke/systemic embolism rates were consistent among older (2.29% rivaroxaban versus 2.85% warfarin per 100 patient-years; hazard ratio=0.80; 95% confidence interval, 0.63-1.02) and younger patients (2.00% versus 2.10%/100 patient-years; hazard ratio=0.95; 95% confidence interval, 0.76-1.19; interaction P=0.313), as were major bleeding rates (≥75 years: 4.86% rivaroxaban versus 4.40% warfarin per 100 patient-years; hazard ratio=1.11; 95% confidence interval, 0.92-1.34; <75 years: 2.69% versus 2.79%/100 patient-years; hazard ratio=0.96; 95% confidence interval, 0.78-1.19; interaction P=0.336). Hemorrhagic stroke rates were similar in both age groups; there was no interaction between age and rivaroxaban response. Elderly patients had higher stroke and major bleeding rates than younger patients, but the efficacy and safety of rivaroxaban relative to warfarin did not differ with age, supporting rivaroxaban as an alternative for the elderly. © 2014 American Heart Association, Inc.

  9. CD-XA Reusable Launch Vehicle (RLV)

    NASA Technical Reports Server (NTRS)

    1995-01-01

    This is the McDornell Douglas CD-XA Reusable Launch Vehicle (RLV) concept. The Delta Clipper-Experimental (DC-X) was originally developed by McDonnell Douglas for the DOD. The DC-XA is a single-stage-to-orbit, vertical takeoff/vertical landing, launch vehicle concept, whose development is geared to significantly reduce launch cost and provided a test bed for NASA Reusable Launch Vehicle (RLV) technology as the Delta Clipper-Experimental Advanced (DC-XA). The program was discontinued in 2003.

  10. Polyphenol Compound as a Transcription Factor Inhibitor

    PubMed Central

    Park, Seyeon

    2015-01-01

    A target-based approach has been used to develop novel drugs in many therapeutic fields. In the final stage of intracellular signaling, transcription factor–DNA interactions are central to most biological processes and therefore represent a large and important class of targets for human therapeutics. Thus, we focused on the idea that the disruption of protein dimers and cognate DNA complexes could impair the transcriptional activation and cell transformation regulated by these proteins. Historically, natural products have been regarded as providing the primary leading compounds capable of modulating protein–protein or protein-DNA interactions. Although their mechanism of action is not fully defined, polyphenols including flavonoids were found to act mostly as site-directed small molecule inhibitors on signaling. There are many reports in the literature of screening initiatives suggesting improved drugs that can modulate the transcription factor interactions responsible for disease. In this review, we focus on polyphenol compound inhibitors against dimeric forms of transcription factor components of intracellular signaling pathways (for instance, c-jun/c-fos (Activator Protein-1; AP-1), c-myc/max, Nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and β-catenin/T cell factor (Tcf)). PMID:26529010

  11. Factors associated with major bleeding events: insights from the ROCKET AF trial (rivaroxaban once-daily oral direct factor Xa inhibition compared with vitamin K antagonism for prevention of stroke and embolism trial in atrial fibrillation).

    PubMed

    Goodman, Shaun G; Wojdyla, Daniel M; Piccini, Jonathan P; White, Harvey D; Paolini, John F; Nessel, Christopher C; Berkowitz, Scott D; Mahaffey, Kenneth W; Patel, Manesh R; Sherwood, Matthew W; Becker, Richard C; Halperin, Jonathan L; Hacke, Werner; Singer, Daniel E; Hankey, Graeme J; Breithardt, Gunter; Fox, Keith A A; Califf, Robert M

    2014-03-11

    This study sought to report additional safety results from the ROCKET AF (Rivaroxaban Once-daily oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation). The ROCKET AF trial demonstrated similar risks of stroke/systemic embolism and major/nonmajor clinically relevant bleeding (principal safety endpoint) with rivaroxaban and warfarin. The risk of the principal safety and component bleeding endpoints with rivaroxaban versus warfarin were compared, and factors associated with major bleeding were examined in a multivariable model. The principal safety endpoint was similar in the rivaroxaban and warfarin groups (14.9 vs. 14.5 events/100 patient-years; hazard ratio: 1.03; 95% confidence interval: 0.96 to 1.11). Major bleeding risk increased with age, but there were no differences between treatments in each age category (<65, 65 to 74, ≥75 years; pinteraction = 0.59). Compared with those without (n = 13,455), patients with a major bleed (n = 781) were more likely to be older, current/prior smokers, have prior gastrointestinal (GI) bleeding, mild anemia, and a lower calculated creatinine clearance and less likely to be female or have a prior stroke/transient ischemic attack. Increasing age, baseline diastolic blood pressure (DBP) ≥90 mm Hg, history of chronic obstructive pulmonary disease or GI bleeding, prior acetylsalicylic acid use, and anemia were independently associated with major bleeding risk; female sex and DBP <90 mm Hg were associated with a decreased risk. Rivaroxaban and warfarin had similar risk for major/nonmajor clinically relevant bleeding. Age, sex, DBP, prior GI bleeding, prior acetylsalicylic acid use, and anemia were associated with the risk of major bleeding. (An Efficacy and Safety Study of Rivaroxaban With Warfarin for the Prevention of Stroke and Non-Central Nervous System Systemic Embolism in Patients With Non-Valvular Atrial Fibrillation: NCT00403767

  12. DOUGLAS XA3D-1 #413 AIRPLANE.

    NASA Image and Video Library

    1955-07-27

    DOUGLAS XA3D-1 #413 AIRPLANE MOUNTED IN THE NACA AMES RESEARCH CENTER'S 40X80_FOOT SUBSONIC WIND TUNNEL Testing the boundary layer control of the A3D in the 40 x 80 wind tunnel. Boundary layer control was added to increase the lift of the wing for take off from an aircraft carrier.

  13. DOUGLAS XA3D-1 #413 AIRPLANE.

    NASA Image and Video Library

    1955-07-27

    DOUGLAS XA3D-1 #413 AIRPLANE MOUNTED IN THE NACA AMES RESEARCH CENTER'S 40X80_FOOT SUBSONIC WIND TUNNEL sweptback wing Testing the wing boundary layer control of the A3D in the 40 x 80 wind tunnel. Boundary layer control was added to increase the lift of the wing for aircraft carrier take off and landing.

  14. High-resolution genetic mapping of rice bacterial blight resistance gene Xa23.

    PubMed

    Wang, Chunlian; Fan, Yinglun; Zheng, Chongke; Qin, Tengfei; Zhang, Xiaoping; Zhao, Kaijun

    2014-10-01

    Bacterial blight (BB) caused by Xanthomonas oryzae pv. oryzae (Xoo) is the most devastating bacterial disease of rice (Oryza sativa L.), a staple food crop that feeds half of the world's population. In management of this disease, the most economical and effective approach is cultivating resistant varieties. Due to rapid change of pathogenicity in the pathogen, it is necessary to identify and characterize more host resistance genes for breeding new resistant varieties. We have previously identified the BB resistance (R) gene Xa23 that confers the broadest resistance to Xoo strains isolated from different rice-growing regions and preliminarily mapped the gene within a 1.7 cm region on the long arm of rice chromosome 11. Here, we report fine genetic mapping and in silico analysis of putative candidate genes of Xa23. Based on F2 mapping populations derived from crosses between Xa23-containing rice line CBB23 and susceptible varieties JG30 or IR24, six new STS markers Lj36, Lj46, Lj138, Lj74, A83B4, and Lj13 were developed. Linkage analysis revealed that the new markers were co-segregated with or closely linked to the Xa23 locus. Consequently, the Xa23 gene was mapped within a 0.4 cm region between markers Lj138 and A83B4, in which the co-segregating marker Lj74 was identified. The corresponding physical distance between Lj138 and A83B4 on Nipponbare genome is 49.8 kb. Six Xa23 candidate genes have been annotated, including four candidate genes encoding hypothetical proteins and the other two encoding a putative ADP-ribosylation factor protein and a putative PPR protein. These results will facilitate marker-assisted selection of Xa23 in rice breeding and molecular cloning of this valuable R gene.

  15. (1)H, (15)N and (13)C resonance assignments of Ixolaris, a tissue factor pathway inhibitor from the tick salivary gland.

    PubMed

    De Paula, V S; Silva, F H S; Francischetti, I M B; Monteiro, R Q; Valente, A P

    2017-08-30

    Ixolaris is a two-Kunitz tick salivary gland protein identified in Ixodes scapularis that presents sequence homology to TFPI (tissue factor pathway inhibitor). It binds to the coagulation enzyme factor Xa (FXa) or to its zymogen form, FX, and further inhibits tissue factor/FVIIa complex (extrinsic Xnase compex). Differently from TFPI, Ixolaris does not bind to the active site cleft of FXa. Instead, complex formation is mediated by the FXa heparin-binding exosite, which may also results in decreased FXa activity into the prothrombinase complex. The Ixolaris-FXa/FX complex formation has been characterized by using a combination of biophysical and biochemical technics although no structural data is currently available. In this study, we reported the NMR chemical shift assignment of Ixolaris, as a first step to further establishing the structure, dynamics and function relationship for this protein.

  16. Mox: a novel modifier of the tomato Xa locus.

    PubMed

    Peterson, P W; Yoder, J I

    1995-01-01

    We have isolated a novel mutation that caused variegated leaf color in a tomato plant which had multiple maize Ac transposable elements and the tomato Xa allele. Xa is a previously characterized semi-dominant mutation that causes tomato leaves to be bright yellow when heterozygous (Xa/xa+). The mutation responsible for the new phenotype was named Mox (Modifier of Xa). The Mox mutation modified the Xa/xa+ yellow leaf phenotype in two ways: it compensated for the Xa allele resulting in a plant with a wildtype green color, and it caused somatic variegation which appeared as white and yellow sectors on the green background. Somatic variegation was visible only if the plant contained both the Mox and Xa loci. Genetic studies indicated that the Mox locus was linked in repulsion to Xa and that the Mox locus was genetically transmitted at a reduced frequency through the male gamete. Molecular characterization of the Ac elements in lines segregating for Mox identified an Ac insertion that appeared to cosegregate with Mox variegation. We propose a model in which the Mox mutation consists of a duplication of the xa+ allele and subsequent Ac-induced breakage of the duplicated region causes variegation.

  17. Proteinase Inhibitor-inducing Factor in Plant Leaves

    PubMed Central

    McFarland, Douglas; Ryan, Clarence A.

    1974-01-01

    Thirty-nine plant species representing 20 families from the four major divisions of plants were surveyed for the presence of proteinase inhibitor-inducing factor activity in leaves or other tissues. Tissue juices were assayed for their capacity to induce accumulation of proteinase inhibitor I in excised tomato (Lycopersico esculentum) leaves. In tissues of only 2 of the 39 species was proteinase inhibitor-inducing factor-like activity not found. The activity was absent in cabbage leaves and celery stalks. Fruiting bodies from one of three fungi genera assayed contained exceptionally large quantities of proteinase inhibitor-inducing factor-like activity. Extracts from Agraricus campestris fruiting bodies contained over 20 times more activity than tomato leaf juice. The survey confirms that substances with proteinase inhibitor-inducing factor-like activity are widespread in the plant kingdom. PMID:16658956

  18. Tumor necrosis factor inhibitors – state of knowledge

    PubMed Central

    Lis, Krzysztof; Kuzawińska, Olga

    2014-01-01

    Tumor necrosis factor (TNF) is considered a major proinflammatory cytokine, affecting various aspects of the immune reaction. All five TNF inhibitors currently available on the market (i.e., etanercept, infliximab, adalimumab, certolizumab and golimumab) are top sellers, although indicated only in autoimmune diseases, including rheumatoid arthritis, Crohn's disease and psoriasis. This article briefly discusses the background and place for TNF inhibitors in modern therapy. The main safety aspects of TNF inhibitor administration are described in particular, with special consideration of the available meta-analyses. Finally, perspectives on the next-generation TNF inhibitors and their use in the clinic are given. PMID:25624856

  19. Astrocyte Mitogen Inhibitor Related to Epidermal Growth Factor Receptor

    NASA Astrophysics Data System (ADS)

    Nieto-Sampedro, Manuel

    1988-06-01

    Epidermal growth factor (EGF) is a well-characterized polypeptide hormone with diverse biological activities, including stimulation of astrocyte division. A soluble astrocyte mitogen inhibitor, immunologically related to the EGF receptor, is present in rat brain. Injury to the brain causes a time-dependent reduction in the levels of this inhibitor and the concomitant appearance of EGF receptor on the astrocyte surface. Intracerebral injection of antibody capable of binding the inhibitor caused the appearance of numerous reactive astrocytes. EGF receptor-related inhibitors may play a key role in the control of glial cell division in both normal and injured brain.

  20. Exactin: A specific inhibitor of Factor X activation by extrinsic tenase complex from the venom of Hemachatus haemachatus

    PubMed Central

    Girish, Vallerinteavide Mavelli; Kini, R. Manjunatha

    2016-01-01

    Unwanted clots lead to heart attack and stroke that result in a large number of deaths. Currently available anticoagulants have some drawbacks including their non-specific actions. Therefore novel anticoagulants that target specific steps in the coagulation pathway are being sought. Here we describe the identification and characterization of a novel anticoagulant protein from the venom of Hemachatus haemachatus (African Ringhals cobra) that specifically inhibits factor X (FX) activation by the extrinsic tenase complex (ETC) and thus named as exactin. Exactin belongs to the three-finger toxin (3FTx) family, with high sequence identity to neurotoxins and low identity to the well-characterized 3FTx anticoagulants-hemextin and naniproin. It is a mixed-type inhibitor of ETC with the kinetic constants, Ki’ and Ki determined as 30.62 ± 7.73 nM and 153.75 ± 17.96 nM, respectively. Exactin does not bind to the active site of factor VIIa and factor Xa based on its weak inhibition (IC50 ≫ 300 μM) to the amidolytic activities of these proteases. Exactin shows exquisite macromolecular specificity to FX activation as compared to factor IX activation by ETC. Exactin thus displays a distinct mechanism when compared to other anticoagulants targeting ETC, with its selective preference to ETC-FX [ES] complex. PMID:27558950

  1. Thrombin Inhibitors from Different Animals

    PubMed Central

    Tanaka-Azevedo, A. M.; Morais-Zani, K.; Torquato, R. J. S.; Tanaka, A. S.

    2010-01-01

    Venous and arterial thromboembolic diseases are still the most frequent causes of death and disability in high-income countries. Clinical anticoagulants are inhibitors of enzymes involved in the coagulation pathway, such as thrombin and factor Xa. Thrombin is a key enzyme of blood coagulation system, activating the platelets, converting the fibrinogen to the fibrin net, and amplifying its self-generation by the activation of factors V, VIII, and XI. Thrombin has long been a target for the development of oral anticoagulants. Furthermore, selective inhibitors of thrombin represent a new class of antithrombotic agents. For these reasons, a number of specific thrombin inhibitors are under evaluation for possible use as antithrombotic drugs. This paper summarizes old and new interests of specific thrombin inhibitors described in different animals. PMID:20976270

  2. Antifactor Xa levels versus activated partial thromboplastin time for monitoring unfractionated heparin.

    PubMed

    Vandiver, Jeremy W; Vondracek, Thomas G

    2012-06-01

    Intravenous unfractionated heparin (UFH) remains an important therapeutic agent, particularly in the inpatient setting, for anticoagulation. Historically, the activated partial thromboplastin time (aPTT) has been the primary laboratory test used to monitor and adjust UFH. The aPTT test has evolved since the 1950s, and the historical goal range of 1.5-2.5 times the control aPTT, which first gained favor in the 1970s, has fallen out of favor due to a high degree of variability in aPTT readings from one laboratory to another, and even from one reagent to another. As a result, it is now recommended that the aPTT goal range be based on a corresponding heparin concentration of 0.2-0.4 unit/ml by protamine titration or 0.3-0.7 unit/ml by antifactor Xa assay. Given that several biologic factors can influence the aPTT independent of the effects of UFH, many institutions have transitioned to monitoring heparin with antifactor Xa levels, rather than the aPTT. Clinical data from the last 10-20 years have begun to show that a conversion from aPTT to antifactor Xa monitoring may offer a smoother dose-response curve, such that levels remain more stable, requiring fewer blood samples and dosage adjustments. Given the minimal increased acquisition cost of the antifactor Xa reagents, it can be argued that the antifactor Xa is a cost-effective method for monitoring UFH. In this review, we discuss the relative advantages and disadvantages of the aPTT, antifactor Xa, and protamine titration tests, and provide a clinical framework to guide practitioners who are seeking to optimize UFH monitoring within their own institutions.

  3. Influence of von Willebrand factor on the reactivity of human factor VIII inhibitors with factor VIII.

    PubMed

    Gensana, M; Altisent, C; Aznar, J A; Casaña, P; Hernández, F; Jorquera, J I; Magallón, M; Massot, M; Puig, L

    2001-07-01

    In order to determine the difference in reactivity of factor (F) VIII inhibitors against the FVIII/von Willebrand factor (vWF) complex and against vWF-deficient FVIII, we investigated a panel of 10 antibodies to FVIII from multitransfused individuals with severe haemophilia A and other pathologies. Immunoblotting of purified FVIII and purified thrombin-cleaved FVIII revealed that in all cases inhibitor epitopes could be localized in the heavy chain (A2 subunit) while in four cases they were also present in the light chain. One of the FVIII inhibitors remained unclassified. The effect on FVIII:C of purified IgG from inhibitor plasmas was tested against a high purity FVIII/vWF concentrate and a monoclonally purified FVIII concentrate with only trace contents of vWF, by two different functional assays. Our results suggest that for those inhibitors showing A2 plus light chain (LC) reactivity, the IgG concentration required to inhibit 50% of FVIII activity in vitro is higher for the FVIII/vWF complex than for the vWF-deficient FVIII. We conclude that there might be a protective role of vWF (at least in vitro) against FVIII inhibitors with A2 and LC subunit specificity.

  4. Rituximab in the treatment of acquired factor VIII inhibitors.

    PubMed

    Wiestner, Adrian; Cho, Hearn J; Asch, Adam S; Michelis, Mary Ann; Zeller, Jack A; Peerschke, Ellinor I B; Weksler, Babette B; Schechter, Geraldine P

    2002-11-01

    Autoantibodies against factor VIII (FVIII) are rare but can cause life-threatening bleeding requiring costly factor replacement and prolonged immunosuppression. We report 4 consecutively treated patients whose acquired FVIII inhibitors responded rapidly to immunosuppressive regimens that included rituximab, a monoclonal antibody against CD20(+) B cells. Three patients had spontaneously occurring inhibitors. The fourth, a patient with mild hemophilia A, developed both an autoantibody and an alloantibody following recombinant FVIII treatment. Pretreatment FVIII activities ranged from less than 1% to 4% and inhibitor titers from 5 to 60 Bethesda units (BU). One patient with polymyalgia rheumatica who developed the inhibitor while receiving prednisone responded to single agent rituximab. The hemophilia patient had rapid resolution of the autoantibody, whereas the alloantibody persisted for months. Responses continue off treatment from more than 7 to more than 12 months. This report adds to the growing evidence that rituximab has efficacy in immune disorders resulting from autoantibody formation.

  5. Membranous nephropathy with acquired factor V inhibitor: a case report

    PubMed Central

    2013-01-01

    Background Membranous nephropathy is one of the most common causes of nephrotic syndrome in adults. In contrast, acquired factor V inhibitor is a rare bleeding disorder. Case presentation A 62-year-old Asian man with a history of cerebral hemorrhage, purpura, eosinophilia and hyper immunoglobulin E syndrome developed proteinuria. The bleeding disorder was diagnosed with acquired factor V inhibitors. A renal biopsy revealed that he suffered from membranous nephropathy with glomerular endothelial damage which is reported to be involved in another factor disorder. After the steroid administration, the coagulation test and proteinuria were improved. Conclusions The presence of factor V inhibitors may have been involved in the development of membranous nephropathy. PMID:24360027

  6. Anthrax lethal factor inhibitors as potential countermeasure of the infection.

    PubMed

    Kumar, B V S Suneel; Malik, Siddharth; Grandhi, Pradeep; Dayam, Raveendra; Sarma, J A R P

    2014-01-01

    Anthrax Lethal Factor (LF) is a zinc-dependent metalloprotease, one of the virulence factor of anthrax infection. Three forms of the anthrax infection have been identified: cutaneous (through skin), gastrointestinal (through alimentary tract), and pulmonary (by inhalation of spores). Anthrax toxin is composed of protective antigen (PA), lethal factor (LF), and edema factor (EF). Protective antigen mediates the entry of Lethal Factor/Edema Factor into the cytosol of host cells. Lethal factor (LF) inactivates mitogen-activated protein kinase kinase inducing cell death, and EF is an adenylyl cyclase impairing host defenses. In the past few years, extensive studies are undertaken to design inhibitors targeting LF. The current review focuses on the small molecule inhibitors targeting LF activity and its structure activity relationships (SAR).

  7. Determination of rivaroxaban in patient’s plasma samples by anti-Xa chromogenic test associated to High Performance Liquid Chromatography tandem Mass Spectrometry (HPLC-MS/MS)

    PubMed Central

    Derogis, Priscilla Bento Matos; Sanches, Livia Rentas; de Aranda, Valdir Fernandes; Colombini, Marjorie Paris; Mangueira, Cristóvão Luis Pitangueira; Katz, Marcelo; Faulhaber, Adriana Caschera Leme; Mendes, Claudio Ernesto Albers; Ferreira, Carlos Eduardo dos Santos; França, Carolina Nunes; Guerra, João Carlos de Campos

    2017-01-01

    Rivaroxaban is an oral direct factor Xa inhibitor, therapeutically indicated in the treatment of thromboembolic diseases. As other new oral anticoagulants, routine monitoring of rivaroxaban is not necessary, but important in some clinical circumstances. In our study a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was validated to measure rivaroxaban plasmatic concentration. Our method used a simple sample preparation, protein precipitation, and a fast chromatographic run. It was developed a precise and accurate method, with a linear range from 2 to 500 ng/mL, and a lower limit of quantification of 4 pg on column. The new method was compared to a reference method (anti-factor Xa activity) and both presented a good correlation (r = 0.98, p < 0.001). In addition, we validated hemolytic, icteric or lipemic plasma samples for rivaroxaban measurement by HPLC-MS/MS without interferences. The chromogenic and HPLC-MS/MS methods were highly correlated and should be used as clinical tools for drug monitoring. The method was applied successfully in a group of 49 real-life patients, which allowed an accurate determination of rivaroxaban in peak and trough levels. PMID:28170419

  8. Determination of rivaroxaban in patient's plasma samples by anti-Xa chromogenic test associated to High Performance Liquid Chromatography tandem Mass Spectrometry (HPLC-MS/MS).

    PubMed

    Derogis, Priscilla Bento Matos; Sanches, Livia Rentas; de Aranda, Valdir Fernandes; Colombini, Marjorie Paris; Mangueira, Cristóvão Luis Pitangueira; Katz, Marcelo; Faulhaber, Adriana Caschera Leme; Mendes, Claudio Ernesto Albers; Ferreira, Carlos Eduardo Dos Santos; França, Carolina Nunes; Guerra, João Carlos de Campos

    2017-01-01

    Rivaroxaban is an oral direct factor Xa inhibitor, therapeutically indicated in the treatment of thromboembolic diseases. As other new oral anticoagulants, routine monitoring of rivaroxaban is not necessary, but important in some clinical circumstances. In our study a high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) method was validated to measure rivaroxaban plasmatic concentration. Our method used a simple sample preparation, protein precipitation, and a fast chromatographic run. It was developed a precise and accurate method, with a linear range from 2 to 500 ng/mL, and a lower limit of quantification of 4 pg on column. The new method was compared to a reference method (anti-factor Xa activity) and both presented a good correlation (r = 0.98, p < 0.001). In addition, we validated hemolytic, icteric or lipemic plasma samples for rivaroxaban measurement by HPLC-MS/MS without interferences. The chromogenic and HPLC-MS/MS methods were highly correlated and should be used as clinical tools for drug monitoring. The method was applied successfully in a group of 49 real-life patients, which allowed an accurate determination of rivaroxaban in peak and trough levels.

  9. Discovery of Phenylglycine Lactams as Potent Neutral Factor VIIa Inhibitors.

    PubMed

    Wurtz, Nicholas R; Parkhurst, Brandon L; Jiang, Wen; DeLucca, Indawati; Zhang, Xiaojun; Ladziata, Vladimir; Cheney, Daniel L; Bozarth, Jeffrey R; Rendina, Alan R; Wei, Anzhi; Luettgen, Joseph M; Wu, Yiming; Wong, Pancras C; Seiffert, Dietmar A; Wexler, Ruth R; Priestley, E Scott

    2016-12-08

    Inhibitors of Factor VIIa (FVIIa), a serine protease in the clotting cascade, have shown strong antithrombotic efficacy in preclinical thrombosis models with minimal bleeding liabilities. Discovery of potent, orally active FVIIa inhibitors has been largely unsuccessful because known chemotypes have required a highly basic group in the S1 binding pocket for high affinity. A recently reported fragment screening effort resulted in the discovery of a neutral heterocycle, 7-chloro-3,4-dihydroisoquinolin-1(2H)-one, that binds in the S1 pocket of FVIIa and can be incorporated into a phenylglycine FVIIa inhibitor. Optimization of this P1 binding group led to the first series of neutral, permeable FVIIa inhibitors with low nanomolar potency.

  10. Tumor necrosis factor (TNF) inhibitor therapy for rheumatoid arthritis.

    PubMed

    Segal, Barbara; Rhodus, Nelson L; Patel, Ketan

    2008-12-01

    Rheumatoid arthritis (RA) is a systemic autoimmune disorder characterized by inflammation involving large and small joints. Systemic manifestations as well as involvement of paraoral tissues contribute to morbidity. Tumor necrosis factor (TNF) plays a central role in RA by amplifying inflammation in multiple pathways that lead to joint destruction. Tumor necrosis factor inhibitors were first licensed for clinical use in 1998; 3 have been approved for the treatment of RA: Iinfliximab, etanercept, and adalimumab. The purpose of this paper is to review the pathogenesis of RA, the state of the art of therapy, and the most current information on the safety and efficacy of TNF inhibitors for treatment of RA.

  11. Anti-xa directed protocol for anticoagulation management in children supported with extracorporeal membrane oxygenation.

    PubMed

    O'Meara, L Carlisle; Alten, Jeffrey A; Goldberg, Kellen G; Timpa, Joseph G; Phillips, Jay; Laney, Debbie; Borasino, Santiago

    2015-01-01

    The optimum heparin monitoring method during extracorporeal membrane oxygenation (ECMO) is unknown. We report a protocol utilizing only anti-factor Xa (anti-Xa) to manage anticoagulation in 22 consecutive ECMO patients. Anti-Xa was monitored with heparin titration every hour until goal 0.4-0.8 IU/ml. Once therapeutic, monitoring was progressively spaced up to every 6 hours. Patients received frequent antithrombin III (ATIII). Extracorporeal membrane oxygenation indications were as follows: 13 cardiorespiratory failures, eight extracorporeal cardiopulmonary resuscitations (ECPRs), and one pulmonary hypertension. Median weight was 4 kg, age 12.5 days, and ECMO duration 88 hours. Survival was 50%. Mean heparin dose was 38 ± 11 unit/kg/hr. Eight patients received no heparin for median 9 hours because of postoperative bleeding. Compared with prior activated clotting time (ACT) protocol, there were 20 fewer blood draws per day to manage anticoagulation, p < 0.001. Only 9% of the anti-Xa levels were outside therapeutic range versus 22% using ACT, p < 0.01. Six patients had bleeding complications, and seven had oxygenator change-out. Change-out was associated with blood product administration and bleeding but not with heparin-free period (p = 0.39). Survival to discharge was higher among those who did not require circuit/oxygenator change-outs, 4/7 versus 7/7 (p < 0.01). Anti-factor Xa-based ECMO heparin management protocol is feasible, decreases blood sampling and heparin infusion adjustments, and does not appear to increase complications.

  12. How to train your inhibitor: Design strategies to overcome resistance to Epidermal Growth Factor Receptor inhibitors.

    PubMed

    Milik, Sandra N; Lasheen, Deena S; Serya, Rabah A T; Abouzid, Khaled A M

    2017-07-18

    Epidermal Growth Factor Receptor (EGFR) stands out as a key player in the development of many cancers. Its dysregulation is associated with a vast number of tumors such as non-small-cell lung cancer, colon cancer, head-and-neck cancer, breast and ovarian cancer. Being implicated in the development of a number of the most lethal cancers worldwide, EGFR has long been considered as a focal target for cancer therapies, ever since the FDA approval of "Gefitinib" in 2003 and up to the last FDA approved small molecule EGFR kinase inhibitor "Osimertinib" in 2015. Studies are still going on to find more efficient EGFR inhibitors due to the continuous emergence of resistance to the current inhibitors. Cancerous cells resist EGFR tyrosine kinase inhibitors (TKIs) through various mechanisms, the most commonly reported ones are the T790M mutation and HER2 amplification. Therefore, tackling EGFR TKIs-resistant tumors through a multi-targeting approach comprising a dual EGFR/HER2 inhibitor that is also capable of inhibiting the mutant T790M EGFR is anticipated to overcome drug resistance. In this review, we will survey the structural aspects of EGFR family and the structure-activity relationship of representative dual EGFR/HER2 inhibitors. To follow, we will discuss the structural aspects of the mutation-driven resistance and various design strategies to overcome it. Finally, we will review the SAR of exemplary irreversible dual EGFR/HER2 inhibitors that can overcome the mutation-driven resistance. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  13. DNA-binding small molecules as inhibitors of transcription factors.

    PubMed

    Leung, Chung-Hang; Chan, Daniel Shiu-Hin; Ma, Victor Pui-Yan; Ma, Dik-Lung

    2013-07-01

    Accumulating evidence implicating the role of aberrant transcription factor signaling in the pathogenesis of various human diseases such as cancer and inflammation has stimulated the development of small molecule ligands capable of targeting transcription factor activity and modulating gene expression. The use of DNA-binding small molecules to selectively inhibit transcription factor-DNA interactions represents one possible approach toward this goal. In this review, we summarize the development of DNA-binding small molecule inhibitors of transcription factors from 2004 to 2011, and their binding mode and therapeutic potential will be discussed. © 2012 Wiley Periodicals, Inc.

  14. A novel allosteric inhibitor of macrophage migration inhibitory factor (MIF).

    PubMed

    Bai, Fengwei; Asojo, Oluwatoyin A; Cirillo, Pier; Ciustea, Mihai; Ledizet, Michel; Aristoff, Paul A; Leng, Lin; Koski, Raymond A; Powell, Thomas J; Bucala, Richard; Anthony, Karen G

    2012-08-31

    Macrophage migration inhibitory factor (MIF) is a catalytic cytokine and an upstream mediator of the inflammatory pathway. MIF has broad regulatory properties, dysregulation of which has been implicated in the pathology of multiple immunological diseases. Inhibition of MIF activity with small molecules has proven beneficial in a number of disease models. Known small molecule MIF inhibitors typically bind in the tautomerase site of the MIF trimer, often covalently modifying the catalytic proline. Allosteric MIF inhibitors, particularly those that associate with the protein by noncovalent interactions, could reveal novel ways to block MIF activity for therapeutic benefit and serve as chemical probes to elucidate the structural basis for the diverse regulatory properties of MIF. In this study, we report the identification and functional characterization of a novel allosteric MIF inhibitor. Identified from a high throughput screening effort, this sulfonated azo compound termed p425 strongly inhibited the ability of MIF to tautomerize 4-hydroxyphenyl pyruvate. Furthermore, p425 blocked the interaction of MIF with its receptor, CD74, and interfered with the pro-inflammatory activities of the cytokine. Structural studies revealed a unique mode of binding for p425, with a single molecule of the inhibitor occupying the interface of two MIF trimers. The inhibitor binds MIF mainly on the protein surface through hydrophobic interactions that are stabilized by hydrogen bonding with four highly specific residues from three different monomers. The mode of p425 binding reveals a unique way to block the activity of the cytokine for potential therapeutic benefit in MIF-associated diseases.

  15. Idiopathic Acquired Hemophilia A with Undetectable Factor VIII Inhibitor

    PubMed Central

    Abt, Nicholas B.; Streiff, Michael B.; Gocke, Christian B.; Kickler, Thomas S.; Lanzkron, Sophie M.

    2014-01-01

    Objective. We present the case of a 73-year-old female, with no family or personal history of a bleeding disorder, who had a classic presentation for acquired hemophilia A. Factor VIII activity was low but detectable and a factor VIII inhibitor was undetectable. Methods. The patient's plasma was comprehensively studied to determine the cause of the acquired coagulopathy. Using the Nijmegen modification of the Bethesda assay, no factor VIII autoantibody was measureable despite varying the incubation time from 1 to 3 hours. Results. The aPTT was prolonged at 46.8 seconds, which did not correct in the 4 : 1 mix but did with 1 : 1 mix. Using a one stage factor VIII activity assay, the FVIII activity was 16% and chromogenic FVIII activity was also 16%. The patient was treated with recombinant FVII and transfusion, significantly reducing bleeding. Long-term therapy was initiated with cyclophosphamide and prednisone with normalization of FVIII activity. Conclusions. Physicians can be presented with the challenging clinical picture of an acquired factor VIII inhibitor without a detectable inhibitor by the Bethesda assay. Standard therapy for an acquired hemophilia A should be considered. PMID:24955264

  16. Factor XIa inhibitors: A review of patent literature

    PubMed Central

    Al-Horani, Rami A.; Desai, Umesh R.

    2016-01-01

    Introduction Anticoagulants are the mainstay for prevention and/or treatment of thrombotic disorders. Each clinically used anticoagulant is associated with significant adverse consequences, especially bleeding. Factor XIa (FXIa), a key factor involved in the amplification of procoagulation signal, has been suggested as a major target for anticoagulant drug discovery because of reduced risk of bleeding. Areas covered Our literature search uncovered dozens of industrial and academic patents on the discovery of novel FXIa/FXI inhibitors. Small peptidomimetics, sulfated glycosaminoglycan mimetics, polypeptides, antisense oligonucleotides, and monoclonal antibodies have been developed as inhibitors of FXIa. Although many agents are in early discovery/development phases, the activity and safety of a few have been evaluated in various animal models and in humans. Expert opinion FXIa is a promising drug target for development of effective anticoagulants with limited bleeding complications. Literature reveals a major trend in the number of patent applications over the last three years. These inhibitors exploit different approaches for target inhibition. Allosteric modulation of FXIa and biosynthetic inhibition of FXI are mechanistically unique. Despite initial results in patients undergoing knee anthroplasty as with antisense oligonucleotides, major advances should be realized, particularly with respect to pharmacokinetics, for FXI/FXIa inhibitors to enter the clinic. PMID:26881476

  17. Plasma tissue factor pathway inhibitor levels as a marker for postoperative bleeding after enoxaparin use in deep vein thrombosis prophylaxis in orthopedics and general surgery.

    PubMed

    Hakki, S I; Fareed, J; Hoppensteadt, D A; Abdullah, H; Camblin, J; Nasseri, A F; Hamadeh, O; Wright, T

    2001-01-01

    Low-molecular-weight heparins (LMWH) are widely used as antithrombotic prophylactic pharmaceutical agents in orthopedic and general surgery. Their antithrombotic characteristics are expressed by plasma mediators such as anti-Xa. anti-IIa, and increased release of tissue factor pathway inhibitor (TFPI) from vascular endothelium. The purpose of this clinical research is to study the relation between plasma levels of these mediators and postoperative bleeding. Forty-one consecutive patients undergoing hip or knee arthroplasty (n = 36) and colectomy (n = 5) received the standard enoxaparin (a LMWH) dose preoperatively (general surgery) or immediately postoperatively (orthopedic surgery). Major bleeding was defined as a postoperative drop of > or = 5 g/dL) of hemoglobin. The authors observed that there was a linear relationship between an increase in free/total TFPI ratio levels and postoperative bleeding. When that ratio increased by >60%, the hemoglobin dropped to >5 g/dL (n = 13). This relationship between free/total TFPI ratio increase and postoperative bleeding was statistically significant (P < 0.001). Those who did not bleed (hemoglobin drop was less than 5 g/dL) (n = 28) had a ratio increase (if any) of less than 50%. However, the authors did not observe any statistical relationship between anti-Xa, anti-IIa, or prothrombin time and postoperative bleeding in patients receiving LMWH for deep vein thrombosis prophylaxis in orthopedic and general surgery patients. The authors recommend a pre- and postoperative ratio level measurement whenever major bleeding is anticipated, as adjustments of LMWH dose or frequency might be necessary.

  18. Plasma tissue factor pathway inhibitor levels as a marker for postoperative bleeding after enoxaparin use in deep vein thrombosis prophylaxis in orthopedics and general surgery.

    PubMed

    Hakki, S I; Fareed, J; Hoppensteadt, D A; Abdullah, H; Camblin, J; Nasseri, A F; Hamadeh, O; Wright, T

    2000-10-01

    Low-molecular-weight heparins (LMWH) are widely used as antithrombotic prophylactic pharmaceutical agents in orthopedic and general surgery. Their antithrombotic characteristics are expressed by plasma mediators such as anti-Xa, anti-IIa, and increased release of tissue factor pathway inhibitor (TFPI) from vascular endothelium. The purpose of this clinical research is to study the relation between plasma levels of these mediators and postoperative bleeding. Forty-one consecutive patients undergoing hip or knee arthroplasty (n = 36) and colectomy (n = 5) received the standard enoxaparin (a LMWH) dose preoperatively (general surgery) or immediately postoperatively (orthopedic surgery). Major bleeding was defined as a postoperative drop of > or = 5 g/dL) of hemoglobin. The authors observed that there was a linear relationship between an increase in free/total TFPI ratio levels and postoperative bleeding. When that ratio increased by > 60%, the hemoglobin dropped to > 5 g/dL (n = 17). This relationship between free/total TFPI ratio increase and postoperative bleeding was statistically significant (P < 0.001). Those who did not bleed (hemoglobin drop was less than 5 g/dL) (n = 24) had a ratio increase (if any) of less than 50%. However, the authors did not observe any statistical relationship between anti-Xa, anti-IIa, or prothrombin time and postoperative bleeding in patients receiving LMWH for deep vein thrombosis prophylaxis in orthopedic and general surgery patients. The authors recommend a pre- and postoperative ratio level measurement whenever major bleeding is anticipated, as adjustments of LMWH dose or frequency might be necessary.

  19. Draft Genome Sequence of Enterobacter cloacae subsp. cloacae Strain 08XA1, a Fecal Bacterium of Giant Pandas

    PubMed Central

    Yan, Yue; Zhao, Chuan-Wu; Zhang, Yi-Zheng; Zhang, Zhi-He; Pan, Guang-Lin; Liu, Wen-Wang; Ma, Qing-Yi; Hou, Rong

    2012-01-01

    Enterobacter cloacae, a common pathogenic bacterium, is a Gram-negative bacillus. We analyzed the draft genome of Enterobacter cloacae subsp. cloacae strain 08XA1 from the feces of a giant panda in China. Genes encoding a β-lactamase and efflux pumps, as well as other factors, have been found in the genome. PMID:23209197

  20. The ATLAS ACS 2-TIMI 51 trial and the burden of missing data: (Anti-Xa Therapy to Lower Cardiovascular Events in Addition to Standard Therapy in Subjects With Acute Coronary Syndrome ACS 2-Thrombolysis In Myocardial Infarction 51).

    PubMed

    Krantz, Mori J; Kaul, Sanjay

    2013-08-27

    Rivaroxaban is a factor Xa inhibitor that was recently reviewed by the Food and Drug Administration as a potential therapy to reduce the risk of recurrent atherothrombotic events in patients with acute coronary syndromes. Approval of this drug would represent a paradigm shift away from dual antiplatelet therapy toward long-term triple antithrombotic therapy. However, to date, no other experimental anticoagulant agent has demonstrated a favorable risk-benefit profile in this population, in part because of the expected increased risk in major bleeding by combining aspirin, a P2Y12 receptor inhibitor, and an anticoagulant. Approvability of rivaroxaban was considered largely on the basis of the ATLAS ACS 2-TIMI 51 (Anti-Xa Therapy to Lower Cardiovascular Events in Addition to Standard Therapy in Subjects With Acute Coronary Syndrome ACS 2-Thrombolysis In Myocardial Infarction 51) trial, which demonstrated a significant reduction in a composite of cardiovascular death, myocardial infarction, and stroke. Although the primary efficacy endpoint was met, a substantial amount of missing data was observed. We discuss the impact of missing data in this trial, its implications for informative censoring of safety events (major bleeding), and implications for future cardiovascular outcomes trials. Copyright © 2013 American College of Cardiology Foundation. Published by Elsevier Inc. All rights reserved.

  1. Discovery and development of anthrax lethal factor metalloproteinase inhibitors.

    PubMed

    Turk, Benjamin E

    2008-02-01

    Anthrax is caused by infection with Bacillus anthracis, a spore forming, rod-shaped, encapsulated gram positive bacteria. The disease manifests itself in distinct ways depending on the route of entry of infective bacterial spores: cutaneous, inhalational, and gastrointestinal. Though rare in humans, inhalational anthrax has become a major concern due to the capacity for spores to be weaponized. The limited success of antibiotic therapy has motivated investigation of complementary therapeutic strategies that target the bacteria's secreted toxin. The zinc-dependent metalloproteinase lethal factor (LF) is a critical component of anthrax toxin and an important potential target for small molecule drugs. In the past few years, a number of approaches have been taken to identify LF inhibitors, from generating conventional metal chelating substrate analogs to random screening of diverse compound libraries. These efforts have produced several different classes of specific nanomolar range inhibitors. Some compounds have fared well in animal models for anthrax toxemia and infection, and these inhibitors and their derivatives may form the basis for future therapies to treat the disease in humans.

  2. International biological standards for coagulation factors and inhibitors.

    PubMed

    Hubbard, Anthony R

    2007-04-01

    The use of international biological standards during the last 30 years has proved extremely successful in promoting global harmonization of estimates between laboratories and methods. Experience has led to the identification of physical criteria essential for standards to be suitable for long-term use. High precision of liquid filling coupled with low residual moisture and oxygen and the use of sealed glass ampoules have been found consistent with homogeneous and stable International Standards (ISs). Most plasma coagulation factors and inhibitors are calibrated in International Units (IU), which are defined as the amount of analyte in 1 mL of normal pooled plasma. Adoption of the IU has provided clarity in the definition of normal and abnormal states and has facilitated dose calculation for replacement therapy. The assay of like-versus-like materials (e.g., concentrate versus concentrate) has been found to improve interlaboratory agreement and there are now both plasma and concentrate ISs available for many coagulation factors and inhibitors. Studies into the assay of recombinant factor VIII have indicated that additional measures, such as modifications to assay methodology, are necessary to reduce interlaboratory variability. This experience may prove valuable in the future, when we have to deal increasingly with the challenges to standardization associated with the products of bioengineering.

  3. Marker-aided Incorporation of Xa38, a Novel Bacterial Blight Resistance Gene, in PB1121 and Comparison of its Resistance Spectrum with xa13 + Xa21.

    PubMed

    Ellur, Ranjith K; Khanna, Apurva; S, Gopala Krishnan; Bhowmick, Prolay K; Vinod, K K; Nagarajan, M; Mondal, Kalyan K; Singh, Nagendra K; Singh, Kuldeep; Prabhu, Kumble Vinod; Singh, Ashok K

    2016-07-11

    Basmati rice is preferred internationally because of its appealing taste, mouth feel and aroma. Pusa Basmati 1121 (PB1121) is a widely grown variety known for its excellent grain and cooking quality in the international and domestic market. It contributes approximately USD 3 billion to India's forex earning annually by being the most traded variety. However, PB1121 is highly susceptible to bacterial blight (BB) disease. A novel BB resistance gene Xa38 was incorporated in PB1121 from donor parent PR114-Xa38 using a modified marker-assisted backcross breeding (MABB) scheme. Phenotypic selection prior to background selection was instrumental in identifying the novel recombinants with maximum recovery of recurrent parent phenome. The strategy was effective in delimiting the linkage drag to <0.5 mb upstream and <1.9 mb downstream of Xa38 with recurrent parent genome recovery upto 96.9% in the developed NILs. The NILs of PB1121 carrying Xa38 were compared with PB1121 NILs carrying xa13 + Xa21 (developed earlier in our lab) for their resistance to BB. Both NILs showed resistance against the Xoo races 1, 2, 3 and 6. Additionally, Xa38 also resisted Xoo race 5 to which xa13 + Xa21 was susceptible. The PB1121 NILs carrying Xa38 gene will provide effective control of BB in the Basmati growing region.

  4. Marker-aided Incorporation of Xa38, a Novel Bacterial Blight Resistance Gene, in PB1121 and Comparison of its Resistance Spectrum with xa13 + Xa21

    PubMed Central

    Ellur, Ranjith K.; Khanna, Apurva; S, Gopala Krishnan.; Bhowmick, Prolay K.; Vinod, K. K.; Nagarajan, M.; Mondal, Kalyan K.; Singh, Nagendra K.; Singh, Kuldeep; Prabhu, Kumble Vinod; Singh, Ashok K.

    2016-01-01

    Basmati rice is preferred internationally because of its appealing taste, mouth feel and aroma. Pusa Basmati 1121 (PB1121) is a widely grown variety known for its excellent grain and cooking quality in the international and domestic market. It contributes approximately USD 3 billion to India’s forex earning annually by being the most traded variety. However, PB1121 is highly susceptible to bacterial blight (BB) disease. A novel BB resistance gene Xa38 was incorporated in PB1121 from donor parent PR114-Xa38 using a modified marker-assisted backcross breeding (MABB) scheme. Phenotypic selection prior to background selection was instrumental in identifying the novel recombinants with maximum recovery of recurrent parent phenome. The strategy was effective in delimiting the linkage drag to <0.5 mb upstream and <1.9 mb downstream of Xa38 with recurrent parent genome recovery upto 96.9% in the developed NILs. The NILs of PB1121 carrying Xa38 were compared with PB1121 NILs carrying xa13 + Xa21 (developed earlier in our lab) for their resistance to BB. Both NILs showed resistance against the Xoo races 1, 2, 3 and 6. Additionally, Xa38 also resisted Xoo race 5 to which xa13 + Xa21 was susceptible. The PB1121 NILs carrying Xa38 gene will provide effective control of BB in the Basmati growing region. PMID:27403778

  5. New insights into the biology of tissue factor pathway inhibitor

    PubMed Central

    MARONEY, S. A.; MAST, A. E.

    2015-01-01

    Summary Tissue factor pathway inhibitor (TFPI) dampens the initiation of blood coagulation by inhibiting two potent procoagulant complexes, tissue factor–factor VIIa (TF–FVIIa) and early forms of prothrombinase. TFPI isoforms, TFPIα and TFPIβ, result from alternative splicing of mRNA, producing distinct C-terminal ends of the two proteins. Both isoforms inhibit TF–FVIIa, but only TFPIα can inhibit early forms of prothrombinase by binding of its positively charged C-terminus with high affinity to the acidic B-domain exosite of FVa, which is generated upon activation by FXa. TFPIα and TFPIβ are produced in cultured human endothelial cells, while platelets contain only TFPIα. Knowledge of the anticoagulant mechanisms and tissue expression patterns of TFPIα and TFPIβ have improved our understanding of the phenotypes observed in different mouse models of TFPI deficiency, the east Texas bleeding disorder, and the development of pharmaceutical agents that block TFPI function to treat hemophilia. PMID:26149025

  6. Treatment of rheumatoid arthritis with tumour necrosis factor inhibitors

    PubMed Central

    Mewar, Devesh; Wilson, Anthony G

    2011-01-01

    Advances in our understanding of the key mediators of chronic inflammation and tissue damage characteristic of rheumatoid arthritis (RA) have resulted in the development of novel therapies primarily targeting pro-inflammatory cytokines. Inhibitors of tumour necrosis factor (TNF) are the most widely used of the biological therapies at present with five different agents currently available; four are based on monoclonal anti-TNF antibodies and a soluble TNF receptor-Fc fusion protein. Long-term use of these molecules has proven to be highly effective in the majority of patients; however, around one-third have a suboptimal response potentially leading to further cartilage and bone damage, furthermore these agents are expensive compared with conventional therapies such as methotrexate. Many recent studies have attempted to identify therapeutic response biomarkers of TNF inhibitors which could be used to improve therapeutic targeting. The presence of rheumatoid factor and anti-cyclic citullinated protein antibodies, present in around 65% of RA patients, are associated with a poorer response to anti-TNF agents. Poorer response is also associated with levels of C-reactive protein and cartilage degradation product at initiation of treatment. Intriguingly, genetic studies of variants of TNF and of genes encoding members of the Toll-like receptors, nuclear factor-kappa B and p38 mitogen-activated protein kinase signalling families have been associated with response to individual anti-TNF agents. Continued advances in technologies such as ultra high throughput sequencing and proteomics should facilitate the discovery of additional biomarkers of response to anti-TNF resulting in improved disease control and quality of life for RA patients and reduced costs for healthcare funders. PMID:21039421

  7. Therapeutic inhibitors of tumor necrosis factor in Crohn's disease.

    PubMed

    Ganesan, Srinivasan; Travis, Simon P L; Ahmad, Tariq; Jazrawi, Riadh

    2002-09-01

    Therapeutic options for patients with refractory ulcerative colitis or Crohn's disease have recently been augmented by the introduction of biological therapies. The pro-inflammatory cytokine, tumor necrosis factor (TNF)-alpha is present in elevated concentrations in patients with inflammatory bowel disease and inhibitors of TNF alpha have proved effective as treatment. Strategies aimed at reducing TNF in patients with Crohn's disease, include the mouse/human chimeric monoclonal antibody, infliximab (Centocor Inc), the humanized monoclonal antibody, CDP-571 (Celltech Group plc), the human recombinant TNF receptor fusion protein, etanercept (Immunex Corp), and thalidomide. New approaches, including the use of soluble TNF receptors, appear promising. This article reviews the evidence of therapeutic inhibition of TNF.

  8. Induction of Xa10-like Genes in Rice Cultivar Nipponbare Confers Disease Resistance to Rice Bacterial Blight.

    PubMed

    Wang, Jun; Tian, Dongsheng; Gu, Keyu; Yang, Xiaobei; Wang, Lanlan; Zeng, Xuan; Yin, Zhongchao

    2017-06-01

    Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae, is one of the most destructive bacterial diseases throughout the major rice-growing regions in the world. The rice disease resistance (R) gene Xa10 confers race-specific disease resistance to X. oryzae pv. oryzae strains that deliver the corresponding transcription activator-like (TAL) effector AvrXa10. Upon bacterial infection, AvrXa10 binds specifically to the effector binding element in the promoter of the R gene and activates its expression. Xa10 encodes an executor R protein that triggers hypersensitive response and activates disease resistance. 'Nipponbare' rice carries two Xa10-like genes in its genome, of which one is the susceptible allele of the Xa23 gene, a Xa10-like TAL effector-dependent executor R gene isolated recently from 'CBB23' rice. However, the function of the two Xa10-like genes in disease resistance to X. oryzae pv. oryzae strains has not been investigated. Here, we designated the two Xa10-like genes as Xa10-Ni and Xa23-Ni and characterized their function for disease resistance to rice bacterial blight. Both Xa10-Ni and Xa23-Ni provided disease resistance to X. oryzae pv. oryzae strains that deliver the matching artificially designed TAL effectors (dTALE). Transgenic rice plants containing Xa10-Ni and Xa23-Ni under the Xa10 promoter provided specific disease resistance to X. oryzae pv. oryzae strains that deliver AvrXa10. Xa10-Ni and Xa23-Ni knock-out mutants abolished dTALE-dependent disease resistance to X. oryzae pv. oryzae. Heterologous expression of Xa10-Ni and Xa23-Ni in Nicotiana benthamiana triggered cell death. The 19-amino-acid residues at the N-terminal regions of XA10 or XA10-Ni are dispensable for their function in inducing cell death in N. benthamiana and the C-terminal regions of XA10, XA10-Ni, and XA23-Ni are interchangeable among each other without affecting their function. Like XA10, both XA10-Ni and XA23-Ni locate to the endoplasmic reticulum (ER) membrane

  9. Lenvatinib in combination with golvatinib overcomes hepatocyte growth factor pathway-induced resistance to vascular endothelial growth factor receptor inhibitor

    PubMed Central

    Nakagawa, Takayuki; Matsushima, Tomohiro; Kawano, Satoshi; Nakazawa, Youya; Kato, Yu; Adachi, Yusuke; Abe, Takanori; Semba, Taro; Yokoi, Akira; Matsui, Junji; Tsuruoka, Akihiko; Funahashi, Yasuhiro

    2014-01-01

    Vascular endothelial growth factor receptor (VEGFR) inhibitors are approved for the treatment of several tumor types; however, some tumors show intrinsic resistance to VEGFR inhibitors, and some patients develop acquired resistance to these inhibitors. Therefore, a strategy to overcome VEGFR inhibitor resistance is urgently required. Recent reports suggest that activation of the hepatocyte growth factor (HGF) pathway through its cognate receptor, Met, contributes to VEGFR inhibitor resistance. Here, we explored the effect of the HGF/Met signaling pathway and its inhibitors on resistance to lenvatinib, a VEGFR inhibitor. In in vitro experiments, addition of VEGF plus HGF enhanced cell growth and tube formation of HUVECs when compared with stimulation by either factor alone. Lenvatinib potently inhibited the growth of HUVECs induced by VEGF alone, but cells induced by VEGF plus HGF showed lenvatinib resistance. This HGF-induced resistance was cancelled when the Met inhibitor, golvatinib, was added with lenvatinib. Conditioned medium from tumor cells producing high amounts of HGF also conferred resistance to inhibition by lenvatinib. In s.c. xenograft models based on various tumor cell lines with high HGF expression, treatment with lenvatinib alone showed weak antitumor effects, but treatment with lenvatinib plus golvatinib showed synergistic antitumor effects, accompanied by decreased tumor vessel density. These results suggest that HGF from tumor cells confers resistance to tumor endothelial cells against VEGFR inhibitors, and that combination therapy using VEGFR inhibitors with Met inhibitors may be effective for overcoming resistance to VEGFR inhibitors. Further evaluation in clinical trials is warranted. PMID:24689876

  10. Lenvatinib in combination with golvatinib overcomes hepatocyte growth factor pathway-induced resistance to vascular endothelial growth factor receptor inhibitor.

    PubMed

    Nakagawa, Takayuki; Matsushima, Tomohiro; Kawano, Satoshi; Nakazawa, Youya; Kato, Yu; Adachi, Yusuke; Abe, Takanori; Semba, Taro; Yokoi, Akira; Matsui, Junji; Tsuruoka, Akihiko; Funahashi, Yasuhiro

    2014-06-01

    Vascular endothelial growth factor receptor (VEGFR) inhibitors are approved for the treatment of several tumor types; however, some tumors show intrinsic resistance to VEGFR inhibitors, and some patients develop acquired resistance to these inhibitors. Therefore, a strategy to overcome VEGFR inhibitor resistance is urgently required. Recent reports suggest that activation of the hepatocyte growth factor (HGF) pathway through its cognate receptor, Met, contributes to VEGFR inhibitor resistance. Here, we explored the effect of the HGF/Met signaling pathway and its inhibitors on resistance to lenvatinib, a VEGFR inhibitor. In in vitro experiments, addition of VEGF plus HGF enhanced cell growth and tube formation of HUVECs when compared with stimulation by either factor alone. Lenvatinib potently inhibited the growth of HUVECs induced by VEGF alone, but cells induced by VEGF plus HGF showed lenvatinib resistance. This HGF-induced resistance was cancelled when the Met inhibitor, golvatinib, was added with lenvatinib. Conditioned medium from tumor cells producing high amounts of HGF also conferred resistance to inhibition by lenvatinib. In s.c. xenograft models based on various tumor cell lines with high HGF expression, treatment with lenvatinib alone showed weak antitumor effects, but treatment with lenvatinib plus golvatinib showed synergistic antitumor effects, accompanied by decreased tumor vessel density. These results suggest that HGF from tumor cells confers resistance to tumor endothelial cells against VEGFR inhibitors, and that combination therapy using VEGFR inhibitors with Met inhibitors may be effective for overcoming resistance to VEGFR inhibitors. Further evaluation in clinical trials is warranted.

  11. Development of the epidermal growth factor receptor inhibitor OSI-774.

    PubMed

    Grünwald, Viktor; Hidalgo, Manuel

    2003-06-01

    The epidermal growth factor receptor (EGFR) is a transmembrane receptor involved in the regulation of a complex array of essential biological processes such as cell proliferation and survival. Dysregulation of the EGFR signaling network has been frequently reported in multiple human cancers and has been associated with the processes of tumor development, growth, proliferation, metastasis, and angiogenesis. Inhibition of the EGFR was associated with antitumor effects in preclinical models. On the basis of these data, therapeutics targeting the EGFR were explored in clinical trials. OSI-774 is a small-molecule selective inhibitor of the EGFR tyrosine kinase. In preclinical studies, OSI-774 inhibited the phosphorylation of the EGFR in a dose-dependent and concentration-dependent manner resulting in cell cycle arrest and induction of apoptosis. In in vivo studies, this agent caused tumor growth inhibition and showed synergistic effects when combined with conventional chemotherapy. Subsequent single-agent phase I studies and phase I studies in combination with chemotherapy showed that the agent has a good safety profile and induced tumor growth inhibition in a substantial number of patients with a variety of different solid tumors. Preliminary reports from phase II studies confirmed the excellent tolerability of OSI-774 and showed encouraging preliminary activity. Phase III studies have either been completed or are ongoing in several tumor types such as lung cancer and pancreatic cancer. In summary, OSI-774 is a novel inhibitor of the EGFR tyrosine kinase that has shown promising activity in initial studies and is currently undergoing full development as an anticancer drug.

  12. Epidermal growth factor receptor tyrosine kinase inhibitors as anticancer agents.

    PubMed

    Ciardiello, F

    2000-01-01

    The epidermal growth factor receptor (EGFR)-driven autocrine growth pathway has been implicated in the development and progression of the majority of the most common human epithelial cancers, making the blockade of this growth pathway a promising anticancer therapeutic strategy. Different approaches have been developed to block EGFR activation and/or function in cancer cells. In the past 15 years, various anti-EGFR blocking monoclonal antibodies (MAb), recombinant proteins containing transforming growth factor-alpha (TGFalpha) or EGF fused to toxins, and tyrosine kinase inhibitors (TKIs) have been generated and their biological and potentially therapeutic properties characterised. One of these agents, MAb IMC-C225, a chimeric human-mouse IgG1 MAb, is the first anti-EGFR agent to enter phase II to III clinical trials in patients with cancer. Several small compounds that block the ligand-induced activation of the EGFR tyrosine kinase have been developed. Among these EGFR-TKIs, various quinazoline-derived agents have been synthesised and have shown promising activity as anticancer agents in preclinical models. ZD1839 ('Iressa'), an anilinoquinazoline, is an orally active, selective EGFR-TKI which is currently under clinical evaluation in phase II to III clinical trials in patients with cancer. Preclinical data for ZD1839 strongly support the possibility of potentiating the antitumour activity of conventional chemotherapy with agents that selectively block the EGFR.

  13. Cell penetrating peptide inhibitors of Nuclear Factor-kappa B

    PubMed Central

    Orange, J. S.; May, M. J.

    2010-01-01

    The nuclear factor kappa B (NF-κB) transcription factors are activated by a range of stimuli including pro-inflammatory cytokines. Active NF-κB regulates the expression of genes involved in inflammation and cell survival and aberrant NF-κB activity plays pathological roles in certain types of cancer and diseases characterized by chronic inflammation. NF-κB signaling is an attractive target for the development of novel anti-inflammatory or anti-cancer drugs and we discuss here how the method of peptide transduction has been used to specifically target NF-κB. Peptide transduction relies on the ability of certain small cell-penetrating peptides (CPPs) to enter cells, and a panel of CPP-linked inhibitors (CPP-Is) has been developed to directly inhibit NF-κB signaling. Remarkably, several of these NF-κB-targeting CPP-Is are effective in vivo and therefore offer exciting potential in the clinical setting. PMID:18668204

  14. Activated protein C resistance testing for factor V Leiden.

    PubMed

    Kadauke, Stephan; Khor, Bernard; Van Cott, Elizabeth M

    2014-12-01

    Activated protein C resistance assays can detect factor V Leiden with high accuracy, depending on the method used. Factor Xa inhibitors such as rivaroxaban and direct thrombin inhibitors including dabigatran, argatroban, and bivalirudin can cause falsely normal results. Lupus anticoagulants can cause incorrect results in most current assays. Assays that include dilution into factor V-deficient plasma are needed to avoid interference from factor deficiencies or elevations, which can arise from a wide variety of conditions such as warfarin, liver dysfunction, or pregnancy. The pros and cons of the currently available assays are discussed. © 2014 Wiley Periodicals, Inc.

  15. Identification of factor inhibitors by diagnostic haemostasis laboratories: a large multi-centre evaluation.

    PubMed

    Favaloro, Emmanuel J; Bonar, Roslyn; Duncan, Elizabeth; Earl, Gail; Low, Joyce; Aboud, Margaret; Just, Sarah; Sioufi, John; Street, Alison; Marsden, Katherine

    2006-07-01

    We have assessed the proficiency of diagnostic haemostasis facilities to correctly identify coagulation factor abnormalities and inhibitors. Forty-two laboratories participating in the external Quality Assurance Program (QAP) conducted by the RCPA agreed to participate and were each sent a set of eight samples (each 3 x 1 ml) for evaluation. They were asked to blind test these samples for the presence or absence of inhibitors, and where identified, to perform further analysis (including specific inhibitor analysis). In order to make the exercise more challenging, in addition to true factor inhibitors, samples were provided that reflected potential pre-analytical variables that might arise and complicate inhibitor detection or lead to false inhibitor identification. In brief, the sample set comprised a true high level factor (F) V inhibitor, a true moderate level FVIII inhibitor (but sample was defibrinogenated), a true lupus anticoagulant (LA), a normal (but slightly aged) plasma sample, a normal serum sample, a normal EDTA sample, an oral anticoagulant/vitamin K deficiency sample, and a gross heparin ( approximately 10 U/ml) contaminated sample. Sixty-three percent of participants correctly identified the true FV inhibitor as such, although the reported range varied greatly [10 to >250 Bethesda units (BU/ml)] and 46% correctly identified the true FVIII inhibitor, despite the complication of the sample presentation, although the reported range also varied (7 to 64 BU/ml). Some laboratories either failed to identify the inhibitor present, or misidentified the inhibitor type. The LA, the oral anticoagulant/vitamin K deficiency, the normal serum sample, and the normal (aged) sample were also correctly identified by most laboratories, as was the absence of specific factor inhibitors in these samples. However, a small subset of laboratories incorrectly identified the presence of specific factor inhibitors in some of these samples. The heparin sample was also correctly

  16. Regulation of factor XIa activity by platelets and alpha 1-protease inhibitor.

    PubMed Central

    Walsh, P N; Sinha, D; Kueppers, F; Seaman, F S; Blankstein, K B

    1987-01-01

    We have studied the complex interrelationships between platelets, Factor XIa, alpha 1-protease inhibitor and Factor IX activation. Platelets were shown to secrete an inhibitor of Factor XIa, and to protect Factor XIa from inactivation in the presence of alpha 1-protease inhibitor and the secreted platelet inhibitor. This protection of Factor XIa did not arise from the binding of Factor XIa to platelets, the presence of high molecular weight kininogen, or the inactivation of alpha 1-protease inhibitor by platelets. The formation of a complex between alpha 1-protease inhibitor and the active-site-containing light chain of Factor XIa was inhibited by activated platelets and by platelet releasates, but not by high molecular weight kininogen. These results support the hypothesis that platelets can regulate Factor XIa-catalyzed Factor IX activation by secreting an inhibitor of Factor XIa that may act primarily outside the platelet microenvironment and by protecting Factor XIa from inhibition, thereby localizing Factor IX activation to the platelet plug. Images PMID:3500185

  17. Proteolytic inactivation of tissue factor pathway inhibitor by bacterial omptins

    PubMed Central

    Yun, Thomas H.; Cott, Jessica E.; Tapping, Richard I.; Slauch, James M.

    2009-01-01

    The immune response to infection includes activation of the blood clotting system, leading to extravascular fibrin deposition to limit the spread of invasive microorganisms. Some bacteria have evolved mechanisms to counteract this host response. Pla, a member of the omptin family of Gram-negative bacterial proteases, promotes the invasiveness of the plague bacterium, Yersinia pestis, by activating plasminogen to plasmin to digest fibrin. We now show that the endogenous anticoagulant tissue factor pathway inhibitor (TFPI) is also highly sensitive to proteolysis by Pla and its orthologs OmpT in Escherichia coli and PgtE in Salmonella enterica serovar Typhimurium. Using gene deletions, we demonstrate that bacterial inactivation of TFPI requires omptin expression. TFPI inactivation is mediated by proteolysis since Western blot analysis showed that TFPI cleavage correlated with loss of anticoagulant function in clotting assays. Rates of TFPI inactivation were much higher than rates of plasminogen activation, indicating that TFPI is a better substrate for omptins. We hypothesize that TFPI has evolved sensitivity to proteolytic inactivation by bacterial omptins to potentiate procoagulant responses to bacterial infection. This may contribute to the hemostatic imbalance in disseminated intravascular coagulation and other coagulopathies accompanying severe sepsis. PMID:18988866

  18. Fibroblast growth factor is an inhibitor of chondrocyte terminal differentiation

    SciTech Connect

    Kato, Y.; Iwamoto, M. )

    1990-04-05

    The effects of basic fibroblast growth factor (bFGF) on terminal differentiation of chondrocytes and cartilage-matrix calcification were investigated. Rabbit growth-plate chondrocytes maintained as a pelleted mass in a centrifuge tube produced an abundant proteoglycan matrix during the matrix-maturation stage, yielding a cartilage-like tissue. Thereafter, they terminally differentiated to hypertrophic chondrocytes which produced high levels of alkaline phosphatase. These cells induced extensive calcification of the matrix in the absence of additional phosphate. Addition of bFGF to the chondrocyte cultures abolished the increases in alkaline phosphatase activity, {sup 45}Ca deposition, and the calcium content. These effects were dose-dependent, reversible, and observed in the presence of cytosine arabinoside, an inhibitor of DNA synthesis. The inhibitory effects could be observed only when chondrocytes were exposed to bFGF in a transition period between the matrix-maturation and hypertrophic stages. As chondrocytes differentiated to hypertrophic cells, bFGF became less effective in inhibiting the expression of the mineralization-related phenotypes. The present study also shows that although the rate of ({sup 35}S)sulfate incorporation into large, chondroitin sulfate proteoglycan in the cell-matrix fraction is very high during the matrix-maturation stage, it abruptly decreases by 90% after terminal differentiation. Furthermore, the terminal differentiation-associated decrease in proteoglycan synthesis was delayed by bFGF. These results provide evidence that bFGF inhibits terminal differentiation of chondrocytes and calcification.

  19. [New anticoagulants - direct thrombin inhibitors].

    PubMed

    Brand, B; Graf, L

    2012-11-01

    Direct thrombin-inhibitors inactivate not only free but also fibrin-bound thrombin. The group of parenteral direct thrombin-inhibitors includes the recombinant hirudins lepirudin and desirudin, the synthetic hirudin bivalirudin, and the small molecule argatroban. All these compounds do not interact with PF4/heparin-antibodies. Therefore, argatroban as well as bivalirudin are currently used to treat heparin-induced thrombocytopenia (HIT). The oral direct thrombin-inhibitor dabigatran etexilate is already licensed in many countries for the treatment of non-valvular atrial fibrillation. Dabigatran etexilate reveals a stable and predictable effect that allows a medication without dose adjustment or monitoring. The substance shows only few interactions with other drugs but strong inhibitors of p-glycoprotein can increase plasma levels of dabigatran substantially. After oral intake, the prodrug dabigatran etexilate is cleaved by esterase-mediated hydrolyses to the active compound dabigatran. Elimination of dabigatran is predominantly renal. Safety and efficacy of dabigatran etexilate were tested in an extensive clinical study program. Non-inferiority compared to current standard treatments was shown for prophylaxis of venous thromboembolic events after total knee and hip replacement, for stroke prevention in atrial fibrillation, and for treatment of acute venous thromboembolism. In daily practice, Dabigatran etexilate competes against the new direct factor Xa-inhibitors. In the absence of direct comparative clinical trials, it is not yet clear if one class of substances has distinct advantages over the other.

  20. Synthesis and P1' SAR exploration of potent macrocyclic tissue factor-factor VIIa inhibitors

    SciTech Connect

    Ladziata, Vladimir; Glunz, Peter W.; Zou, Yan; Zhang, Xiaojun; Jiang, Wen; Jacutin-Porte, Swanee; Cheney, Daniel L.; Wei, Anzhi; Luettgen, Joseph M.; Harper, Timothy M.; Wong, Pancras C.; Seiffert, Dietmar; Wexler, Ruth R.; Priestley, E. Scott

    2016-10-01

    Selective tissue factor-factor VIIa complex (TF-FVIIa) inhibitors are viewed as promising compounds for treating thrombotic disease. In this contribution, we describe multifaceted exploratory SAR studies of S1'-binding moieties within a macrocyclic chemotype aimed at replacing cyclopropyl sulfone P1' group. Over the course of the optimization efforts, the 1-(1H-tetrazol-5-yl)cyclopropane P1' substituent emerged as an improved alternative, offering increased metabolic stability and lower clearance, while maintaining excellent potency and selectivity.

  1. Edoxaban vs warfarin in patients with nonvalvular atrial fibrillation in the US Food and Drug Administration approval population: An analysis from the Effective Anticoagulation with Factor Xa Next Generation in Atrial Fibrillation-Thrombolysis in Myocardial Infarction 48 (ENGAGE AF-TIMI 48) trial.

    PubMed

    Eisen, Alon; Giugliano, Robert P; Ruff, Christian T; Nordio, Francesco; Gogia, Harinder S; Awasty, Vivek R; Henderson, David A; Mercuri, Michele F; Rutman, Howard; Antman, Elliott M; Braunwald, Eugene

    2016-02-01

    Edoxaban is a specific anti-Xa inhibitor that, in comparison to warfarin, has been found to be noninferior for the prevention of stroke or systemic embolism (SSE) and to reduce bleeding significantly in patients with nonvalvular atrial fibrillation (AF). The US Food and Drug Administration (FDA) approved the higher-dose edoxaban regimen (60/30 mg) in patients with AF and a creatinine clearance of ≤95 mL/min. We report for the first time the clinical characteristics, efficacy, and safety of the FDA-approved population in the ENGAGE AF--TIMI 48 trial. The patients included had been treated with either warfarin or edoxaban 60/30 mg and had a creatinine clearance of ≤95 mL/min. The primary efficacy was SSE, and the principal safety end point was major bleeding (International Society on Thrombosis and Haemostasis classification). Median follow-up was 2.8 years. Patients in the FDA-approved cohort were older, were more likely female, and had higher CHADS2 and HAS-BLED scores, as compared with patients not included in the FDA label. The primary end point occurred in 1.63%/y with edoxaban vs 2.02%/y with warfarin (hazard ratio [HR] 0.81, 95% CI 0.67-0.97, P = .023). Edoxaban significantly reduced the rate of hemorrhagic stroke (HR 0.47, 95% CI 0.31-0.72, P < .001) and cardiovascular death (HR 0.84, 95% CI 0.73-0.97, P = .015). Ischemic stroke rates were similar between the treatment groups (1.31%/y vs 1.39%/y, P = .97). Major bleeding was significantly lower with edoxaban (3.16%/y vs 3.77%/y; HR 0.84, 95% CI 0.72-0.98, P = .023). In the FDA-approved cohort of the ENGAGE AF--TIMI 48 trial, treatment with edoxaban 60/30 mg was superior to warfarin in the prevention of SSE and significantly reduced cardiovascular death and bleeding, especially fatal bleeding and hemorrhagic stroke. Copyright © 2015 Elsevier Inc. All rights reserved.

  2. Vasculitis Associated With Tumor Necrosis FactorInhibitors

    PubMed Central

    Sokumbi, Olayemi; Wetter, David A.; Makol, Ashima; Warrington, Kenneth J.

    2012-01-01

    Objective To describe the clinical characteristics, histopathologic features, and outcomes of patients in whom vasculitis developed in association with use of tumor necrosis factor-α (TNF-α) inhibitors. Patients and Methods This is a retrospective review of patients evaluated at Mayo Clinic, Rochester, Minnesota, from January 1, 1998, through March 31, 2011, with a diagnosis of vasculitis induced by anti–TNF-α therapy. Results Of 8 patients with vasculitis associated with anti–TNF-α therapy (mean age, 48.5 years), 6 (75%) were female. Four (50%) had rheumatoid arthritis, 1 (13%) had Crohn disease, and 3 (38%) had ulcerative colitis. Five (63%) were treated with infliximab, 2 (25%) with etanercept, and 1 (13%) with adalimumab. The mean duration of treatment before development of vasculitis was 34.5 months. The skin was the predominant organ affected (5 patients [63%]), with the most common cutaneous lesion being palpable purpura (4 of 5 [80%]). Two organs involved in systemic vasculitis were the peripheral nervous system (4 patients [50%]) and kidney (1 patient [13%]). All cases of vasculitis were histopathologically confirmed. Seven of 8 patients improved with discontinuation of therapy (mean time to resolution, 6.9 months) and adjuvant treatment (all 8 received prednisone; another agent was also used in 7); rechallenge with anti–TNF-α therapy was not attempted in any patient. At last follow-up, no patients had experienced a recurrence of vasculitis after therapy discontinuation. Conclusion Cutaneous small-vessel vasculitis was the most common finding, but systemic vasculitis, including peripheral nerve and renal vasculitis, was also frequently observed. PMID:22795634

  3. Analysis of the influence of dabigatran on coagulation factors and inhibitors.

    PubMed

    Tsutsumi, Y; Shimono, J; Ohhigashi, H; Ito, S; Shiratori, S; Teshima, T

    2015-04-01

    Dabigatran is an oral intake thrombin inhibitor for preventive administration against stroke accompanied by atrial fibrillation. Although dabigatran causes prolonged activated partial thromboplastin time (APTT), the effect of dabigatran on each coagulation factor and coagulation factor inhibitor remains to be investigated. Our aim was to analyze the influence of dabigatran on coagulation factors and coagulation factor inhibitors. We administered dabigatran to 40 patients. In 26 of these 40, we analyzed the activity of several coagulation factors and their inhibitors. We used Fisher's exact test to determine statistical significance. The activities of many coagulation factors changed during the dabigatran therapy. Factor II levels decreased in all patients showing prolongation of partial thromboplastin (PT) and APTT. The antifactor VIII inhibitor was positive in the majority of patients with prolonged PT and APTT, while activities of protein C, protein S, and antifactor IX inhibitor were not associated with PT and APTT prolongation. Dabigatran affects the activities of many coagulation factors, including factors II, V, VIII, and IX, as well as the antifactor VIII inhibitor. © 2014 John Wiley & Sons Ltd.

  4. Inactivation of factor XII active fragment in normal plasma. Predominant role of C-1-inhibitor.

    PubMed Central

    de Agostini, A; Lijnen, H R; Pixley, R A; Colman, R W; Schapira, M

    1984-01-01

    To define the factors responsible for the inactivation of the active fragment derived from Factor XII (Factor XIIf ) in plasma, we studied the inactivation kinetics of Factor XIIf in various purified and plasma mixtures. We also analyzed the formation of 125I-Factor XIIf -inhibitor complexes by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In purified systems, the bimolecular rate constants for the reactions of Factor XIIf with C-1-inhibitor, alpha 2-antiplasmin, and antithrombin III were 18.5, 0.91, and 0.32 X 10(4) M-1 min-1, respectively. Furthermore, SDS-PAGE analysis revealed that 1:1 stoichiometric complexes were formed between 125I-Factor XIIf and each of these three inhibitors. In contrast, kinetic and SDS-PAGE studies indicated that Factor XIIf did not react with alpha 1-antitrypsin or alpha 2-macroglobulin. The inactivation rate constant of Factor XIIf by prekallikrein-deficient plasma was 14.4 X 10(-2) min-1, a value that was essentially identical to the value predicted from the studies in purified systems (15.5 X 10(-2) min-1). This constant was reduced to 1.8 X 10(-2) min-1 when Factor XIIf was inactivated by prekallikrein-deficient plasma that had been immunodepleted (less than 5%) of C-1-inhibitor. In addition, after inactivation in normal plasma, 74% of the active 125I-Factor XIIf was found to form a complex with C-1-inhibitor, whereas 26% of the enzyme formed complexes with alpha 2-antiplasmin and antithrombin III. Furthermore, 42% of the labeled enzyme was still complexed with C-1-inhibitor when 125I-Factor XII was inactivated in hereditary angioedema plasma that contained 32% of functional C-1-inhibitor. This study quantitatively demonstrates the dominant role of C-1-inhibitor in the inactivation of Factor XIIf in the plasma milieu. Images PMID:6725552

  5. XA21-specific induction of stress-related genes following Xanthomonas infection of detached rice leaves

    PubMed Central

    Liu, Furong; Chen, Huamin; Wei, Tong; Nguyen, Yen P.; Shaker, Isaac W.F.

    2016-01-01

    The rice XA21 receptor kinase confers robust resistance to the bacterial pathogen Xanthomonas oryzaepv. oryzae (Xoo). We developed a detached leaf infection assay to quickly and reliably measure activation of the XA21-mediated immune response using genetic markers. We used RNA sequencing of elf18 treated EFR:XA21:GFP plants to identify candidate genes that could serve as markers for XA21 activation. From this analysis, we identified eight genes that are up-regulated in both in elf18 treated EFR:XA21:GFP rice leaves and Xoo infected XA21 rice leaves. These results provide a rapid and reliable method to assess bacterial-rice interactions. PMID:27703843

  6. Digoxin use in patients with atrial fibrillation and adverse cardiovascular outcomes: a retrospective analysis of the Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF).

    PubMed

    Washam, Jeffrey B; Stevens, Susanna R; Lokhnygina, Yuliya; Halperin, Jonathan L; Breithardt, Günter; Singer, Daniel E; Mahaffey, Kenneth W; Hankey, Graeme J; Berkowitz, Scott D; Nessel, Christopher C; Fox, Keith A A; Califf, Robert M; Piccini, Jonathan P; Patel, Manesh R

    2015-06-13

    Digoxin is a widely used drug for ventricular rate control in patients with atrial fibrillation (AF), despite a scarcity of randomised trial data. We studied the use and outcomes of digoxin in patients in the Rivaroxaban Once Daily Oral Direct Factor Xa Inhibition Compared with Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF). For this retrospective analysis, we included and classified patients from ROCKET AF on the basis of digoxin use at baseline and during the study. Patients in ROCKET AF were recruited from 45 countries and had AF and risk factors putting them at moderate-to-high risk of stroke, with or without heart failure. We used Cox proportional hazards regression models adjusted for baseline characteristics and drugs to investigate the association of digoxin with all-cause mortality, vascular death, and sudden death. ROCKET AF was registered with ClinicalTrials.gov, number NCT00403767. In 14,171 randomly assigned patients, digoxin was used at baseline in 5239 (37%). Patients given digoxin were more likely to be female (42% vs 38%) and have a history of heart failure (73% vs 56%), diabetes (43% vs 38%), and persistent AF (88% vs 77%; p<0·0001 for each comparison). After adjustment, digoxin was associated with increased all-cause mortality (5·41 vs 4·30 events per 100 patients-years; hazard ratio 1·17; 95% CI 1·04-1·32; p=0·0093), vascular death (3·55 vs 2·69 per 100 patient-years; 1·19; 1·03-1·39, p=0·0201), and sudden death (1·68 vs 1·12 events per 100 patient-years; 1·36; 1·08-1·70, p=0·0076). Digoxin treatment was associated with a significant increase in all-cause mortality, vascular death, and sudden death in patients with AF. This association was independent of other measured prognostic factors, and although residual confounding could account for these results, these data show the possibility of digoxin having these effects. A randomised trial of digoxin in treatment of AF patients

  7. A ΩXaV motif in the Rift Valley fever virus NSs protein is essential for degrading p62, forming nuclear filaments and virulence

    PubMed Central

    Cyr, Normand; de la Fuente, Cynthia; Lecoq, Lauriane; Guendel, Irene; Chabot, Philippe R.; Kehn-Hall, Kylene; Omichinski, James G.

    2015-01-01

    Rift Valley fever virus (RVFV) is a single-stranded RNA virus capable of inducing fatal hemorrhagic fever in humans. A key component of RVFV virulence is its ability to form nuclear filaments through interactions between the viral nonstructural protein NSs and the host general transcription factor TFIIH. Here, we identify an interaction between a ΩXaV motif in NSs and the p62 subunit of TFIIH. This motif in NSs is similar to ΩXaV motifs found in nucleotide excision repair (NER) factors and transcription factors known to interact with p62. Structural and biophysical studies demonstrate that NSs binds to p62 in a similar manner as these other factors. Functional studies in RVFV-infected cells show that the ΩXaV motif is required for both nuclear filament formation and degradation of p62. Consistent with the fact that the RVFV can be distinguished from other Bunyaviridae-family viruses due to its ability to form nuclear filaments in infected cells, the motif is absent in the NSs proteins of other Bunyaviridae-family viruses. Taken together, our studies demonstrate that p62 binding to NSs through the ΩXaV motif is essential for degrading p62, forming nuclear filaments and enhancing RVFV virulence. In addition, these results show how the RVFV incorporates a simple motif into the NSs protein that enables it to functionally mimic host cell proteins that bind the p62 subunit of TFIIH. PMID:25918396

  8. Reduction of Factor VIII Inhibitor Titers During Immune Tolerance Induction With Recombinant Factor VIII-Fc Fusion Protein.

    PubMed

    Groomes, Charles L; Gianferante, David M; Crouch, Gary D; Parekh, Dina S; Scott, David W; Lieuw, Kenneth

    2016-05-01

    The development of inhibitors toward factor VIII (FVIII) is a common and serious complication of hemophilia A (HA) therapy. Patients with hemophilia who develop inhibitors often undergo time- and resource-intensive immune tolerance induction (ITI) protocols. We report a 15-month-old male with severe HA and a high-titer inhibitor that occurred while receiving prophylactic treatment with recombinant FVIII (rFVIII), in whom significant inhibitor titer reduction was achieved with thrice weekly infusions of a new, prolonged half-life rFVIII-Fc fusion protein product (trade name Eloctate). Further studies are warranted to explore the potential of Eloctate in ITI protocols.

  9. Genetic factors influencing inhibitor development in a cohort of South African haemophilia A patients.

    PubMed

    Lochan, A; Macaulay, S; Chen, W C; Mahlangu, J N; Krause, A

    2014-09-01

    A critical complication of factor VIII (FVIII) replacement therapy in Haemophilia A (HA) treatment is inhibitor development. Known genetic factors predisposing to inhibitor development include FVIII (F8) gene mutations, ethnicity, a family history of inhibitors and FVIII haplotype mismatch. The aim of this study was to characterize and correlate these genetic factors in a cohort of South African HA patients. This was a retrospective study that included 229 patients and involved the analysis of patient files, HA molecular and clinical databases and molecular analysis of the F8 gene haplotype. Of the 229 patients, 51% were of black ethnicity, 49% were white, 5% had mild HA, 4% were moderate and 91% were severe, 36% were int22 positive and 13% were inhibitor positive. Of the inhibitor positive patients, 72% were black patients. Inhibitors were reported in 27% of black int22 positive patients, 13% of black int22 negative patients, 9% of white int22 positive patients and 7% of white int22 negative. The H1 haplotype was more common in whites (75%) and H2 was more common in blacks (74%). H3 and H5 were only found in black patients and had a higher frequency of inhibitor development than H1 and H2. In this small HA cohort, black patients had a significantly higher frequency of inhibitor development and the results were indicative of an association between inhibitor development, ethnicity and haplotype.

  10. Laboratory identification of factor VIII inhibitors in the real world: the experience from Australasia.

    PubMed

    Favaloro, E J; Bonar, R; Kershaw, G; Mohammed, S; Duncan, E; Marsden, K

    2010-07-01

    The laboratory has a key role in the initial detection of factor inhibitors and an ongoing role in the measurement of inhibitor titres during the course of inhibitor eradication therapy. The most commonly seen factor inhibitors are those directed against factor VIII (FVIII), usually detected either using the original or Nijmegen-modified Bethesda assay. In view of previously demonstrated high variability in laboratory results for inhibitor assays, we have more extensively examined laboratory performance in the identification of FVIII inhibitors. Over the past 3 years, we conducted two questionnaire-based surveys and two wet-challenge surveys utilizing eight samples comprising no FVIII inhibitor (n = 1), or low-titre (n = 2), medium-titre (n = 3) or high-titre (n = 2) FVIII inhibitor. Four samples were tested by 42 laboratories in 2007, and four by 52 laboratories in 2009. High inter-laboratory variation was evident, with CVs around 50% not uncommon, and some 10% of all laboratories (or around 15% of laboratories using Bethesda method) failed to detect low-level inhibitors of around 1 BU mL(-1). Laboratories using the Nijmegen method appeared to perform better than those using a standard Bethesda assay, with lower evident assay variation and no false negatives. There was a wide variety of laboratory practice, with no two laboratories using exactly the same process for testing and interpretation of factor inhibitor findings. In conclusion, our study indicates that there is still much need for standardization and improvement in factor inhibitor detection, and we hope that our findings provide a basis for future improvements in this area.

  11. The prevalence of factor VIII and IX inhibitors among Saudi patients with hemophilia

    PubMed Central

    Owaidah, Tarek; Momen, Abdulkareem Al; Alzahrani, Hazzaa; Almusa, Abdulrahman; Alkasim, Fawaz; Tarawah, Ahmed; Nouno, Randa Al; Batniji, Fatima Al; Alothman, Fahad; Alomari, Ali; Abu-Herbish, Saud; Abu-Riash, Mahmoud; Siddiqui, Khawar; Ahmed, Mansor; Mohamed, SY; Saleh, Mahasen

    2017-01-01

    Abstract Hemophilia A and B are X-linked diseases that predominantly affect male patients. Patients can develop coagulation factor inhibitors, which exponentially increases the treatment cost. However, the prevalence of factor VIII and IX inhibitors in Saudi Arabia is unclear. This study aimed to determine the Saudi prevalence of factor VIII and IX inhibitors. This 4-year, 7-center, cross-sectional study evaluated the Saudi prevalences of hemophilia A and B. We collected the patients’ clinical data, evaluated their disease, and tested for factor inhibitors. We included 202 patients with hemophilia (median age at diagnosis: 0.13 years, range: birth–34.8 years). The patients included 198 male patients (98%), 148 patients with hemophilia A (73.3%), and 54 patients with hemophilia B (26.7%). The patients exhibited severe factor VIII activity (<1%; 121 patients; 5.2%), moderate activity (1–5%; 7 patients; 4.9%), and mild activity (14 patients; 9.9%). Among the patients with care-related data, most patients were treated for episodic bleeding (76.8%) or received prophylaxis (22.6%); 1 patient received both treatments. Among the patients with source-related data, the factor replacements were derived from plasma (48.4%), recombinant concentrates (22.9%), both sources (14.6%), or fresh frozen plasma (14.1%). Factor VIII inhibitors were observed in 43 (29.3%) of the 147 patients, and only 1 of the 54 patients developed factor IX inhibitors. Most patients who developed inhibitors had severe hemophilia (40/44; 90.9%), and inhibitors were also common among patients who received recombinant products (14/43; 32.6%). The Saudi prevalence of factor inhibitors was similar to those among other ethnic populations. PMID:28079788

  12. Treatment of patients with hemophilia A and inhibitors to factor FVIII with cimetidine.

    PubMed

    Ambriz Fernandez, R; Quintana Gonzalez, S; Martinez Murillo, C; Dominguez Garcia, V; Rodriguez Moyado, H; Collazo Jaloma, J

    1996-01-01

    In this study, cimetidine was used to treat patients with hemophilia A and inhibitors to factor VIII who presented with acute hemorrhages (Group A) and those without hemorrhages (Group B). The dose of cimetidine was 15 mg/kg/day. Group A consisted of five patients with inhibitors between 156 and > 10,000 Bethesda Units (BU), all with serious hemorrhagic problems. The control of hemorrhaging was effective in 100% of these patients, although inhibitor levels remained high (25-380 BU). Group B consisted of seven patients who did not have hemorrhages, whose inhibitor levels were 41-358 BU. Five of these patients no longer had anamnestic responses to Factor VIII after several months of treatment with cimetidine. No difference in the response to cimetidine was seen between HIV positive and HIV negative patients. The results suggest that cimetidine is useful to suppress inhibitors to Factor VIII in patients with hemophilia A.

  13. Comparison of three different anti-Xa assays in major orthopedic surgery patients treated with fondaparinux.

    PubMed

    Ikejiri, Makoto; Wada, Hideo; Yamaguchi, Toshio; Miyazaki, Shinichi; Hasegawa, Masahiro; Wakabayashi, Hiroki; Asanuma, Kunihiro; Sakaguchi, Akane; Matsumoto, Takeshi; Ohishi, Kohshi; Fujimoto, Naoki; Yamada, Norikazu; Ito, Masaaki; Katayama, Naoyuki; Sudo, Akihiro

    2016-05-01

    Anti-Xa assays are useful for monitoring the effects of selective anti-Xa drugs, such as fondaparinux, in the prophylaxis of deep vein thrombosis. In the present study, anti-Xa activity was measured using three different assays, Testzym(®) Heparin S, STA(®)-Liquid Anti-Xa and HemosIL(®) Liquid Heparin. Anti-Xa activity in each assay gradually increased from day one after administration to day eight, and still remained on day 15. Although there were significant differences in anti-Xa activity among the three assays, the activity showed significant correlation across assays. There were no significant differences in the anti-Xa activity between patients with and without DVT or between patients with and without massive bleeding on day one before and after administration, day four, day eight and day 15. Anti-Xa activity in each assay was weakly correlated with antithrombin (AT) activity. The AT activity in patients were significantly higher on days four, eight and 15 compared with day one before and after administration, suggesting that AT activity increases following the administration of fondaparinux. The three anti-Xa assay kits tested are useful for monitoring fondaparinux treatment in orthopedic surgery patients.

  14. Rice Xa21 primed genes and pathways that are critical for combating bacterial blight infection.

    PubMed

    Peng, Hai; Chen, Zheng; Fang, Zhiwei; Zhou, Junfei; Xia, Zhihui; Gao, Lifen; Chen, Lihong; Li, Lili; Li, Tiantian; Zhai, Wenxue; Zhang, Weixiong

    2015-07-17

    Rice bacterial blight (BB) is a devastating rice disease. The Xa21 gene confers a broad and persistent resistance against BB. We introduced Xa21 into Oryza sativa L ssp indica (rice 9311), through multi-generation backcrossing, and generated a nearly isogenic, blight-resistant 9311/Xa21 rice. Using next-generation sequencing, we profiled the transcriptomes of both varieties before and within four days after infection of bacterium Xanthomonas oryzae pv. oryzae. The identified differentially expressed (DE) genes and signaling pathways revealed insights into the functions of Xa21. Surprisingly, before infection 1,889 genes on 135 of the 316 signaling pathways were DE between the 9311/Xa21 and 9311 plants. These Xa21-mediated basal pathways included mainly those related to the basic material and energy metabolisms and many related to phytohormones such as cytokinin, suggesting that Xa21 triggered redistribution of energy, phytohormones and resources among essential cellular activities before invasion. Counter-intuitively, after infection, the DE genes between the two plants were only one third of that before the infection; other than a few stress-related pathways, the affected pathways after infection constituted a small subset of the Xa21-mediated basal pathways. These results suggested that Xa21 primed critically important genes and signaling pathways, enhancing its resistance against bacterial infection.

  15. Protease inhibitors interfere with the necessary factors of carcinogenesis.

    PubMed

    Troll, W

    1989-05-01

    Many tumor promoters are inflammatory agents that stimulate the formation of oxygen radicals (.O2-) and hydrogen peroxide (H2O2) in phagocytic neutrophils. The neutrophils use the oxygen radicals to kill bacteria, which are recognized by the cell membrane of phagocytic cells causing a signal to mount the oxygen response. The tumor promoter isolated from croton oil, 12-O-tetradecanoylphorbol-13-acetate (TPA), mimics the signal, causing an oxygen radical release that is intended to kill bacteria; instead, it injures cells in the host. Oxygen radicals cause single strand breaks in DNA and modify DNA bases. These damaging reactions appear to be related to tumor promotion, as three types of chemopreventive agents, retinoids, onion oil, and protease inhibitors, suppress the induction of oxygen radicals in phagocytic neutrophils and suppress tumor promotion in skin cancer in mice. Protease inhibitors also suppress breast and colon cancers in mice. Protease inhibitors capable of inhibiting chymotrypsin show a greater suppression of the oxygen effect and are better suppressors of tumor promotion. In addition, oxygen radicals may be one of the many agents that cause activation of oncogenes. Since retinoids and protease inhibitors suppress the expression of the ras oncogene in NIH 3T3 cells, NIH 3T3 cells may serve as a relatively facile model for finding and measuring chemopreventive agents that interfere with the carcinogenic process.

  16. Evaluation of aspirin metabolites as inhibitors of hypoxia-inducible factor hydroxylases.

    PubMed

    Lienard, Benoit M; Conejo-García, Ana; Stolze, Ineke; Loenarz, Christoph; Oldham, Neil J; Ratcliffe, Peter J; Schofield, Christopher J

    2008-12-21

    Known and potential aspirin metabolites were evaluated as inhibitors of oxygen-sensing hypoxia-inducible transcription factor (HIF) hydroxylases; some of the metabolites were found to stabilise HIF-alpha in cells.

  17. Hemodialysis in a patient with severe hemophilia A and factor VIII inhibitor.

    PubMed

    Gopalakrishnan, Natarajan; Usha, Thiruvengadam; Thopalan, Balasubramaniyan; Dhanapriya, Jeyachandran; Dineshkumar, Thanigachalam; Thirumalvalavan, Kaliaperumal; Sakthirajan, Ramanathan

    2016-10-01

    Hemophilia A is a hereditary X-linked recessive disease caused by mutations in the gene encoding factor VIII (FVIII), occurring in 1 out of 10,000 persons. Life expectancy and quality of life have dramatically improved recently in patients with hemophilia. Chronic kidney disease and need for renal replacement therapy in these patients are rare. The development of inhibitors to FVIII is the most serious complication of hemophilia and makes treatment of bleeds very challenging. We describe here a 28-year-old male patient with severe hemophilia A with presence of factor VIII inhibitor, who had end stage renal disease. Central venous access device was inserted along with infusion of factor eight inhibitor bypass activity before and after the procedure. He is currently on thrice weekly hemodialysis and doing well for 6 months without bleeding episodes. To our knowledge, hemophilia A with factor VIII inhibitor managed with hemodialysis has not been reported so far. © 2016 International Society for Hemodialysis.

  18. Pattern of factor VIII inhibitors in patients with hemophilia A in the north east of Iran.

    PubMed

    Modaresi, A R; Torghabeh, H Mansouri; Pourfathollah, A A; Shooshtari, M Mahmoodian; Yazdi, Z Rezaie

    2006-06-01

    This survey was conducted to evaluate coagulation factor VIII:C inhibitors among 102 hemophilia A patients from different cities of Khorasan province in north east of Iran in order to identify and characterize the pattern of inhibitor formation in these patients population. For this purpose, we randomly obtained plasma samples of 102 hemophilia A patients (44 patients with severe, 28 patients with intermediate and 30 patients with mild hemophilia A) and studied them using two tests: the APTT mix and Bethesda test were performed. In the whole group 20 patients (19.6%) factor VIII inhibitors were detected. These were in 11 patients with severe, five patients with intermediate and four patients with mild hemophilia A. None of patients with hemophilia A had previously been studied for the presence of an inhibitor, so there was no existing history of inhibitor evaluation.

  19. Outcomes of temporary interruption of rivaroxaban compared with warfarin in patients with nonvalvular atrial fibrillation: results from the rivaroxaban once daily, oral, direct factor Xa inhibition compared with vitamin K antagonism for prevention of stroke and embolism trial in atrial fibrillation (ROCKET AF).

    PubMed

    Sherwood, Matthew W; Douketis, James D; Patel, Manesh R; Piccini, Jonathan P; Hellkamp, Anne S; Lokhnygina, Yuliya; Spyropoulos, Alex C; Hankey, Graeme J; Singer, Daniel E; Nessel, Christopher C; Mahaffey, Kenneth W; Fox, Keith A A; Califf, Robert M; Becker, Richard C

    2014-05-06

    During long-term anticoagulation in atrial fibrillation, temporary interruptions (TIs) of therapy are common, but the relationship between patient outcomes and TIs has not been well studied. We sought to determine reasons for TI, the characteristics of patients undergoing TI, and the relationship between anticoagulant and outcomes among patients with TI. In the Rivaroxaban Once Daily, Oral, Direct Factor Xa Inhibition Compared With Vitamin K Antagonism for Prevention of Stroke and Embolism Trial in Atrial Fibrillation (ROCKET AF), a randomized, double-blind, double-dummy study of rivaroxaban and warfarin in nonvalvular atrial fibrillation, baseline characteristics, management, and outcomes, including stroke, non-central nervous system systemic embolism, death, myocardial infarction, and bleeding, were reported in participants who experienced TI (3-30 days) for any reason. The at-risk period for outcomes associated with TI was from TI start to 30 days after resumption of study drug. In 14 236 participants who received at least 1 dose of study drug, 4692 (33%) experienced TI. Participants with TI were similar to the overall ROCKET AF population in regard to baseline clinical characteristics. Only 6% (n=483) of TI incidences involved bridging therapy. Stroke/systemic embolism rates during the at-risk period were similar in rivaroxaban-treated and warfarin-treated participants (0.30% versus 0.41% per 30 days; hazard ratio [confidence interval]=0.74 [0.36-1.50]; P=0.40). Risk of major bleeding during the at-risk period was also similar in rivaroxaban-treated and warfarin-treated participants (0.99% versus 0.79% per 30 days; hazard ratio [confidence interval]=1.26 [0.80-2.00]; P=0.32). TI of oral anticoagulation is common and is associated with substantial stroke risks and bleeding risks that were similar among patients treated with rivaroxaban or warfarin. Further investigation is needed to determine the optimal management strategy in patients with atrial fibrillation

  20. Structures of adamalysin II with peptidic inhibitors. Implications for the design of tumor necrosis factor alpha convertase inhibitors.

    PubMed Central

    Gomis-Rüth, F. X.; Meyer, E. F.; Kress, L. F.; Politi, V.

    1998-01-01

    Crotalus adamanteus snake venom adamalysin II is the structural prototype of the adamalysin or ADAM family comprising proteolytic domains of snake venom metalloproteinases, multimodular mammalian reproductive tract proteins, and tumor necrosis factor alpha convertase, TACE, involved in the release of the inflammatory cytokine, TNFalpha. The structure of adamalysin II in noncovalent complex with two small-molecule right-hand side peptidomimetic inhibitors (Pol 647 and Pol 656) has been solved using X-ray diffraction data up to 2.6 and 2.8 A resolution. The inhibitors bind to the S'-side of the proteinase, inserting between two protein segments, establishing a mixed parallel-antiparallel three-stranded beta-sheet and coordinate the central zinc ion in a bidentate manner via their two C-terminal oxygen atoms. The proteinase-inhibitor complexes are described in detail and are compared with other known structures. An adamalysin-based model of the active site of TACE reveals that these small molecules would probably fit into the active site cleft of this latter metalloproteinase, providing a starting model for the rational design of TACE inhibitors. PMID:9521103

  1. Mutation of the rice XA21 predicted nuclear localization sequence does not affect resistance to Xanthomonas oryzae pv. oryzae

    DOE PAGES

    Wei, Tong; Chen, Tsung-Chi; Ho, Yuen Ting; ...

    2016-10-05

    Background: The rice receptor kinase XA21 confers robust resistance to the bacterial pathogenXanthomonas oryzae pv.oryzae(Xoo). We previously reported that XA21 is cleaved in transgenic plants overexpressing XA21 with a GFP tag (Ubi-XA21-GFP) and that the released C-terminal domain is localized to the nucleus. XA21 carries a predicted nuclear localization sequence (NLS) that directs the C-terminal domain to the nucleus in transient assays, whereas alanine substitutions in the NLS disrupt the nuclear localization. Methods: To determine if the predicted NLS is required for XA21-mediated immunityin planta, we generated transgenic plants overexpressing an XA21 variant carrying the NLS with the same alaninemore » substitutions (Ubi-XA21nls-GFP). Results: Ubi- XA21nls-GFP plants displayed slightly longer lesion lengths, higherXoo bacterial populations after inoculation and lower levels of reactive oxygen species production compared with the Ubi- XA21-GFP control plants. However, the Ubi- XA21nls-GFP plants express lower levels of protein than that observed inUbi- XA21-GFP. Discussion: These results demonstrate that the predicted NLS is not required for XA21-mediated immunity.« less

  2. A close insight to factor VIII inhibitor in the congenital hemophilia A.

    PubMed

    Tabriznia-Tabrizi, Shamsoreza; Gholampour, Marzie; Mansouritorghabeh, Hassan

    2016-09-01

    Hemophilia A (HA) has an X-linked pattern of inheritance and is the most common of the hemorrhagic disorders. HA is caused by a decreased or deficiency of the functional clotting factor VIII (FVIII) and effects 1 in 5000-10,000 male births. The common treatment for hemophilia is replacement therapy by plasma-derived or recombinant FVIII. Approximately 20-30% of people with a severe type of HA develop an inhibitor and this phenomenon is the main challenge in the management of these patients. Genetic factors and environmental determinants contribute to inhibitor development. Here, the roles of various genetic and environmental factors such as the type of FVIII concentrate used, the number of exposure days, and peak treatment time will be discussed in detail. It seems this information is helpful for hematologists. A literature review was done in January 2016 on PubMed and Scopus using the following keywords:' h(a)emophilia A & factor VIII inhibitor', 'h(a)emophilia A & factor VIII alloantibody', 'h(a)emophilia A & inhibitor'. There was no time limitation; however, there was an English language limitation placed on the articles selected. Expert commentary: Influential genetic and environmental factors in developing inhibitors have been discussed. Most of the risk factors are related to previously untreated patients with hemophili.

  3. Clinical experience of anti-Xa monitoring in critically ill dogs receiving dalteparin.

    PubMed

    Lynch, Alex M; deLaforcade, Armelle M; Sharp, Claire R

    2014-01-01

    To describe a population of critically ill dogs receiving dalteparin monitored with an anti-Xa assay, to assess the potential utility of serial monitoring, and to investigate the association between pre-treatment thromboelastography (TEG) and the ability to achieve targeted anti-Xa activity. Descriptive retrospective study. Veterinary teaching hospital. Thirty-eight client-owned dogs receiving dalteparin and monitored with an anti-Xa assay. None. Medical records were retrospectively reviewed for signalment, underlying disease, clinicopathological data, occurrence of thromboembolic events, complications, and outcome. Thirty-eight dogs receiving dalteparin were monitored with an anti-Xa assay. Diseases included hematological disease, protein-losing disease, neoplastic disease, and septic processes. Pretreatment hypercoagulability was present in 34/35 dogs by assessment of TEG. Five cases of thromboembolism were confirmed prior to starting treatment and 4 cases occurred during hospitalization. Bleeding complications were rare (3/38) and 29/38 dogs survived to discharge. Interpretation of the anti-Xa assay allowed for dose adjustment although reliable achievement of target anti-Xa activity was not demonstrated. Dogs with higher G values on pretreatment TEG were significantly less likely to achieve the target anti-Xa activity (ie, be above or below the target range). Dalteparin was well tolerated in a heterogeneous population of dogs. However, dose adjustment in response to anti-Xa activity interpretation inconsistently resulted in subsequent attainment of the target anti-Xa range. Development of guidelines may be warranted to more consistently achieve the target range. Dogs that appear more hypercoagulable on pre-treatment TEG may require closer monitoring and greater dose adjustment to achieve the target anti-Xa range. © Veterinary Emergency and Critical Care Society 2014.

  4. An evaluation of risk factors for adverse drug events associated with angiotensin-converting enzyme inhibitors.

    PubMed

    Morimoto, Takeshi; Gandhi, Tejal K; Fiskio, Julie M; Seger, Andrew C; So, Joseph W; Cook, E Francis; Fukui, Tsuguya; Bates, David W

    2004-11-01

    To identify potential factors leading to discontinuation of angiotensin-converting enzyme (ACE) inhibitors because of adverse drug events. Retrospective cohort study was conducted at outpatient clinics affiliated with an urban tertiary care hospital. ACE inhibitors were administered to 2225 consecutive outpatients. In 19% of the total cohort, ACE inhibitors were discontinued because of adverse drug events. Cox proportional hazard model identified the following independent risk factors for discontinuation because of adverse drug events: age, female gender, ethnicity other than African American or Latino, no history of previous ACE inhibitor use, history of cough caused by another ACE inhibitor, hypertension, anxiety or depression, no hemodialysis, and elevated creatinine. History of smoking was shown to be a risk factor for cough [hazard ratio (HR): 2.5; 95% confidence interval (CI): 1.1-5.7], angioedema (HR: 2.7; 95% CI: 1.1-7.0), and hyperkalaemia (HR: 5.4; 95% CI: 1.3-23.2). History of ACE inhibitor-induced cough was not only a risk factor for cough (HR: 12.9; 95% CI: 7.5-22.3) but also for angioedema (HR: 9.1; 95% CI: 2.1-39.9). Patients with creatinine > or = 1.6 mg dL(-1) were likely to discontinue ACE inhibitors because of renal dysfunction (HR: 4.7; 95% CI: 1.5-12.7) and hyperkalaemia (HR: 10.9; 95% CI: 3.1-39.0). East Asians were more likely to develop cough (HR: 2.5; 95% CI: 1.1-5.7) and hyperkalaemia (HR: 80.3; 95% CI: 5.4-1190) and African Americans to develop angioedema (HR: 3.5; 95% CI: 1.3-8.9). Although further validation is necessary, these risk factors should help doctors identify patients with elevated risk for adverse drug events because of ACE inhibitors.

  5. Molecular phylogeny, homology modeling, and molecular dynamics simulation of race-specific bacterial blight disease resistance protein (xa5) of rice: a comparative agriproteomics approach.

    PubMed

    Dehury, Budheswar; Sahu, Mousumi; Sarma, Kishore; Sahu, Jagajjit; Sen, Priyabrata; Modi, Mahendra Kumar; Sharma, Gauri Dutta; Choudhury, Manabendra Dutta; Barooah, Madhumita

    2013-08-01

    Rice (Oryza sativa L.), a model plant belonging to the family Poaceae, is a staple food for a majority of the people worldwide. Grown in the tropical and subtropical regions of the world, this important cereal crop is under constant and serious threat from both biotic and abiotic stresses. Among the biotic threats, Xanthomonas oryzae pv. oryzae, causing the damaging bacterial blight disease in rice, is a prominent pathogen. The xa5 gene in the host plant rice confers race-specific resistance to this pathogen. This recessive gene belongs to the Xa gene family of rice and encodes a gamma subunit of transcription factor IIA (TFIIAγ). In view of the importance of this gene in conferring resistance to the devastating disease, we reconstructed the phylogenetic relationship of this gene, developed a three-dimensional protein model, followed by long-term molecular dynamics simulation studies to gain a better understanding of the evolution, structure, and function of xa5. The modeled structure was found to fit well with the small subunit of TFIIA from human, suggesting that it may also act as a small subunit of TFIIA in rice. The model had a stable conformation in response to the atomic flexibility and interaction, when subjected to MD simulation at 20 nano second in aqueous solution. Further structural analysis of xa5 indicated that the protein retained its basic transcription factor function, suggesting that it might govern a novel pathway responsible for bacterial blight resistance. Future molecular docking studies of xa5 underway with its corresponding avirulence gene is expected to shed more direct light into plant-pathogen interactions at the molecular level and thus pave the way for richer agriproteomic insights.

  6. Sulfated Pentagalloylglucoside is a Potent, Allosteric, and Selective Inhibitor of Factor XIa

    PubMed Central

    Al-Horani, Rami A.; Ponnusamy, Pooja; Mehta, Akul Y.; Gailani, David; Desai, Umesh R.

    2013-01-01

    Inhibition of factor XIa (FXIa) is a novel paradigm for developing anticoagulants without major bleeding consequences. We present the discovery of sulfated pentagalloylglucoside (6) as a highly selective inhibitor of human FXIa. Biochemical screening of a focused library led to the identification of 6, a sulfated aromatic mimetic of heparin. Inhibitor 6 displayed a potency of 551 nM against FXIa, which was at least 200-fold more selective than other relevant enzymes. It also prevented activation of factor IX and prolonged human plasma and whole blood clotting. Inhibitor 6 reduced VMAX of FXIa hydrolysis of chromogenic substrate without affecting the KM suggesting an allosteric mechanism. Competitive studies showed that 6 bound in the heparin-binding site of FXIa. No allosteric small molecule has been discovered to date that exhibits equivalent potency against FXIa. Inhibitor 6 is expected to open up a major route to allosteric FXIa anticoagulants with clinical relevance. PMID:23316863

  7. Acquired factor V inhibitor after exposure to topical human thrombin related to an otorhinolaryngological procedure.

    PubMed

    Donohoe, K; Levine, R

    2015-10-01

    Acquired factor V (FV) inhibitors occur rarely and classically develop after exposure to bovine thrombin. The clinical presentation is variable, ranging from asymptomatic with incidental laboratory abnormalities to significant bleeding. With the development of human-derived thrombin agents, bovine thrombin is less frequently used. We report a case of an acquired FV inhibitor that developed in a patient after exposure to human thrombin used as a hemostatic agent during an otorhinolaryngology surgical procedure. Our review of the literature revealed only one prior reported case of FV inhibitor after exposure to human thrombin. Hematologists and surgeons should be aware of this rare, but potentially life-threatening, complication in the postprocedural setting.

  8. What factors determine patients' preference for tumour necrosis factor inhibitors in ankylosing spondylitis?

    PubMed

    Fajri, Dessy W; Brand, Caroline A; Dharmage, Shyamali C; Martin, Belinda J; Buchanan, Russell R C; Schachna, Lionel

    2009-05-01

    Tumour necrosis factor inhibitor (TNFi) therapy, either intravenous (IV) or subcutaneous (SQ), demonstrates similar efficacy in ankylosing spondylitis (AS). The objective of this study was to examine factors influencing patient preference of TNFi. Fifty-nine (79.7%) participants were male with mean age 43.9 years and disease duration of 22.0 years. Fifty-nine patients (79.7%) agreed with the statement 'My doctor gave me a choice and I made a decision based on my personal preference'. Patients commenced first on IV TNFi most commonly cited reduced frequency of injections (96.6%), administration by a trained professional (89.7%) and use of infusion time for leisure activities (86.2%). Patients commenced on SQ TNFi cited flexibility with timing of treatment (80%), shortened administration time (73.3%) and the convenience of home therapy (73.3%). Shared clinical decision-making between clinicians and patients may be desirable for AS patients commencing TNFi therapy.

  9. Effect of food, an antacid, and the H2 antagonist ranitidine on the absorption of BAY 59-7939 (rivaroxaban), an oral, direct factor Xa inhibitor, in healthy subjects.

    PubMed

    Kubitza, Dagmar; Becka, Michael; Zuehlsdorf, Michael; Mueck, Wolfgang

    2006-05-01

    To investigate the influence of food and administration of an antacid (aluminum-magnesium hydroxide) or ranitidine on the absorption of BAY 59-7939 (rivaroxaban), 4 randomized studies were performed in healthy male subjects. In 2 food interaction studies, subjects received BAY 59-7939, either as two 5-mg tablets (fasted and fed), four 5-mg tablets (fasted), or one 20-mg tablet (fasted and fed). In 2 drug interaction studies, BAY 59-7939 (six 5-mg tablets) was given alone or with ranitidine (150 mg twice daily, preceded by a 3-day pretreatment phase) or antacid (10 mL). Plasma samples were obtained to assess pharmacokinetic and pharmacodynamic parameters of BAY 59-7939. In the presence of food, time to maximum concentration (t(max)) was delayed by 1.25 hours; maximum concentration (C(max)) and area under the curve (AUC) were increased, with reduced interindividual variability at higher doses of BAY 59-7939. Compared with baseline, BAY 59-7939 resulted in a relative increase in maximum prothrombin time (PT) prolongation of 44% (10 mg) and 53% (20 mg) in the fasted state, compared with 53% and 83% after food. Time to maximum PT prolongation was delayed by 0.5 to 1.5 hours after food, with no relevant influence of food type. No significant difference in C(max) and AUC was observed with coadministration of BAY 59-7939 and ranitidine or antacid.

  10. Bleeding associated with acquired factor V inhibitor in a patient on warfarin treated successfully with prednisolone

    PubMed Central

    Khalafallah, Alhossain; Grabek, Julian; Hayes, Robert; Mohamed, Muhajir

    2013-01-01

    An 85-year-old man on warfarin for atrial fibrillation presented with skin bleeding. International normalised ratio (INR) and activated partial thromboplastin time (APTT) were elevated and did not correct even after warfarin reversal with vitamin K, prothrombin complex concentrate (PCC) and fresh frozen plasma. Mixing coagulation studies with normal plasma suggested the presence of an inhibitor rather than the multiple coagulation factor deficiencies expected with warfarin. Assays of the common-pathway coagulation factors revealed factor V concentration <2% with inhibitor level elevated to 11 Bethesda units. The bleeding resolved following a course of corticosteroids. Coagulation studies and factor V level returned to normal along with resolution of the inhibitor. We report the case of the diagnostic dilemma posed and successful therapy implemented despite the limited evidence-based data being available for the treatment of this rare condition. PMID:23921688

  11. Renal dysfunction as a predictor of stroke and systemic embolism in patients with nonvalvular atrial fibrillation: validation of the R(2)CHADS(2) index in the ROCKET AF (Rivaroxaban Once-daily, oral, direct factor Xa inhibition Compared with vitamin K antagonism for prevention of stroke and Embolism Trial in Atrial Fibrillation) and ATRIA (AnTicoagulation and Risk factors In Atrial fibrillation) study cohorts.

    PubMed

    Piccini, Jonathan P; Stevens, Susanna R; Chang, YuChiao; Singer, Daniel E; Lokhnygina, Yuliya; Go, Alan S; Patel, Manesh R; Mahaffey, Kenneth W; Halperin, Jonathan L; Breithardt, Günter; Hankey, Graeme J; Hacke, Werner; Becker, Richard C; Nessel, Christopher C; Fox, Keith A A; Califf, Robert M

    2013-01-15

    We sought to define the factors associated with the occurrence of stroke and systemic embolism in a large, international atrial fibrillation (AF) trial. In ROCKET AF (Rivaroxaban Once-daily, oral, direct factor Xa inhibition Compared with vitamin K antagonism for prevention of stroke and Embolism Trial in Atrial Fibrillation), 14 264 patients with nonvalvular AF and creatinine clearance ≥30 mL/min were randomized to rivaroxaban or dose-adjusted warfarin. Cox proportional hazards modeling was used to identify factors at randomization independently associated with the occurrence of stroke or non-central nervous system embolism based on intention-to-treat analysis. A risk score was developed in ROCKET AF and validated in ATRIA (AnTicoagulation and Risk factors In Atrial fibrillation), an independent AF patient cohort. Over a median follow-up of 1.94 years, 575 patients (4.0%) experienced primary end-point events. Reduced creatinine clearance was a strong, independent predictor of stroke and systemic embolism, second only to prior stroke or transient ischemic attack. Additional factors associated with stroke and systemic embolism included elevated diastolic blood pressure and heart rate, as well as vascular disease of the heart and limbs (C-index 0.635). A model that included creatinine clearance (R(2)CHADS(2)) improved net reclassification index by 6.2% compared with CHA(2)DS(2)VASc (C statistic=0.578) and by 8.2% compared with CHADS(2) (C statistic=0.575). The inclusion of creatinine clearance <60 mL/min and prior stroke or transient ischemic attack in a model with no other covariates led to a C statistic of 0.590.Validation of R(2)CHADS(2) in an external, separate population improved net reclassification index by 17.4% (95% confidence interval, 12.1%-22.5%) relative to CHADS(2). In patients with nonvalvular AF at moderate to high risk of stroke, impaired renal function is a potent predictor of stroke and systemic embolism. Stroke risk stratification in patients

  12. Factor VIII gene (F8) mutation and risk of inhibitor development in nonsevere hemophilia A.

    PubMed

    Eckhardt, Corien L; van Velzen, Alice S; Peters, Marjolein; Astermark, Jan; Brons, Paul P; Castaman, Giancarlo; Cnossen, Marjon H; Dors, Natasja; Escuriola-Ettingshausen, Carmen; Hamulyak, Karly; Hart, Daniel P; Hay, Charles R M; Haya, Saturnino; van Heerde, Waander L; Hermans, Cedric; Holmström, Margareta; Jimenez-Yuste, Victor; Keenan, Russell D; Klamroth, Robert; Laros-van Gorkom, Britta A P; Leebeek, Frank W G; Liesner, Ri; Mäkipernaa, Anne; Male, Christoph; Mauser-Bunschoten, Evelien; Mazzucconi, Maria G; McRae, Simon; Meijer, Karina; Mitchell, Michael; Morfini, Massimo; Nijziel, Marten; Oldenburg, Johannes; Peerlinck, Kathelijne; Petrini, Pia; Platokouki, Helena; Reitter-Pfoertner, Sylvia E; Santagostino, Elena; Schinco, Piercarla; Smiers, Frans J; Siegmund, Berthold; Tagliaferri, Annarita; Yee, Thynn T; Kamphuisen, Pieter Willem; van der Bom, Johanna G; Fijnvandraat, Karin

    2013-09-12

    Neutralizing antibodies (inhibitors) toward factor VIII form a severe complication in nonsevere hemophilia A, profoundly aggravating the bleeding pattern. Identification of high-risk patients is hampered by lack of data that take exposure days to therapeutic factor VIII concentrates into account. In the INSIGHT study, we analyzed the association between F8 mutation and inhibitor development in patients with nonsevere hemophilia A (factor VIII 2-40 IU/dL). This analysis included 1112 nonsevere hemophilia A patients from 14 centers in Europe and Australia that had genotyped at least 70% of their patients. Inhibitor risk was calculated as Kaplan-Meier incidence with cumulative number of exposure days as the time variable. During 44 800 exposure days (median, 24 exposure days per patient; interquartile range [IQR], 7-90), 59 of the 1112 patients developed an inhibitor; cumulative incidence of 5.3% (95% confidence interval [CI], 4.0-6.6) after a median of 28 exposure days (IQR, 12-71). The inhibitor risk at 50 exposure days was 6.7% (95% CI, 4.5-8.9) and at 100 exposure days the risk further increased to 13.3% (95% CI, 9.6-17.0). Among a total of 214 different F8 missense mutations 19 were associated with inhibitor development. These results emphasize the importance of F8 genotyping in nonsevere hemophilia A.

  13. Factor 8 (F8) gene mutation profile of Turkish hemophilia A patients with inhibitors.

    PubMed

    Fidanci, Inanç D; Kavakli, Kaan; Uçar, Canan; Timur, Cetin; Meral, Adalet; Kilinç, Yurdanur; Sayilan, Hülya; Kazanci, Elif; Cağlayan, S Hande

    2008-07-01

    Factor VIII (FVIII) replacement therapy is ineffective in hemophilia A patients who develop alloantibodies (inhibitors) against FVIII. The type of factor 8 (F8) gene mutation, genes in the major histocompatibility complex loci, and also polymorphisms in IL-10 and tumor necrosis factor-alpha are the major predisposing factors for inhibitor formation. The present study was initiated to reveal the F8 gene mutation profile of 30 severely affected high-responder patients with inhibitor levels of more than 5 Bethesda U (BU)/ml and four low-responder patients with inhibitors less than 5 BU/ml. Southern blot and PCR analysis were performed to detect intron 22 and intron 1 inversions, respectively. Point mutations were screened by DNA sequence analysis of all coding regions, intron/exon boundaries, promoter and 3' UTR regions of the F8 gene. The prevalent mutation was the intron 22 inversion among the high-responder patients followed by large deletions, small deletions, and nonsense mutations. Only one missense and one splicing error mutation was seen. Among the low-responder patients, three single nucleotide deletions and one intron 22 inversion were found. All mutation types detected were in agreement with the severe hemophilia A phenotype, most likely leading to a deficiency of and predisposition to the development of alloantibodies against FVIII. It is seen that Turkish hemophilia A patients with major molecular defects have a higher possibility of developing inhibitors.

  14. Arginine mimetic structures in biologically active antagonists and inhibitors.

    PubMed

    Masic, Lucija Peterlin

    2006-01-01

    Peptidomimetics have found wide application as bioavailable, biostable, and potent mimetics of naturally occurring biologically active peptides. L-Arginine is a guanidino group-containing basic amino acid, which is positively charged at neutral pH and is involved in many important physiological and pathophysiological processes. Many enzymes display a preference for the arginine residue that is found in many natural substrates and in synthetic inhibitors of many trypsin-like serine proteases, e.g. thrombin, factor Xa, factor VIIa, trypsin, and in integrin receptor antagonists, used to treat many blood-coagulation disorders. Nitric oxide (NO), which is produced by oxidation of L-arginine in an NADPH- and O(2)-dependent process catalyzed by isoforms of nitric oxide synthase (NOS), exhibits diverse roles in both normal and pathological physiologies and has been postulated to be a contributor to the etiology of various diseases. Development of NOS inhibitors as well as analogs and mimetics of the natural substrate L-arginine, is desirable for potential therapeutic use and for a better understanding of their conformation when bound in the arginine binding site. The guanidino residue of arginine in many substrates, inhibitors, and antagonists forms strong ionic interactions with the carboxylate of an aspartic acid moiety, which provides specificity for the basic amino acid residue in the active side. However, a highly basic guanidino moiety incorporated in enzyme inhibitors or receptor antagonists is often associated with low selectivity and poor bioavailability after peroral application. Thus, significant effort is focused on the design and preparation of arginine mimetics that can confer selective inhibition for specific trypsin-like serine proteases and NOS inhibitors as well as integrin receptor antagonists and possess reduced basicity for enhanced oral bioavailability. This review will describe the survey of arginine mimetics designed to mimic the function of the

  15. Relation of factor VIII and IX inhibitors with ABO blood groups in 150 patients with haemophilia A and B.

    PubMed

    Torghabeh, Hassan Mansouri; Pourfathollah, Aliakbar; Shooshtari, Mahmood Mahmoodian; Yazdi, Zahra Rezaie

    2006-03-01

    Many investigations have proved relations between ABO blood groups with some diseases and factor VIII and von willebrand level in plasma. In this study we investigated a relation between ABO blood groups and factor VIII and IX inhibitors in 102 patients with haemophilia A and 48 patients with haemophilia B. The assay of inhibitor was done by Bethesda method. There were no relation between ABO blood groups and factor VIII and IX inhibitors.

  16. Antidotes to anthrax lethal factor intoxication. Part 1: Discovery of potent lethal factor inhibitors with in vivo efficacy.

    PubMed

    Jiao, Guan-Sheng; Kim, Seongjin; Moayeri, Mahtab; Cregar-Hernandez, Lynne; McKasson, Linda; Margosiak, Stephen A; Leppla, Stephen H; Johnson, Alan T

    2010-11-15

    Sub-nanomolar small molecule inhibitors of anthrax lethal factor have been identified using SAR and Merck L915 (4) as a model compound. One of these compounds (16) provided 100% protection in a rat lethal toxin model of anthrax disease.

  17. Effect of tumor necrosis factorinhibitors on ambulatory 24-h blood pressure.

    PubMed

    Grossman, Chagai; Bornstein, Gil; Leibowitz, Avshalom; Ben-Zvi, Ilan; Grossman, Ehud

    2017-02-01

    Tumor necrosis factor alpha (TNF-α) inhibitors are increasingly being used in inflammatory rheumatic diseases (IRD). The risk of cardiovascular disease is elevated in patients with IRD and TNF-α inhibitors reduce this risk. We assessed whether the beneficial effect of TNF-α inhibitors on cardiovascular risk is mediated by blood pressure reduction. We measured blood pressure levels with 24-h ambulatory blood pressure measurements device in patients with IRD before and 3 months after treatment with TNF-α inhibitors. The study population consisted of 15 subjects (6 men; mean age 45.9 ± 14.1 years). Most patients had either rheumatoid arthritis or psoriatic arthritis and adalimumab was the most common TNF-α inhibitor used. Mean 24-h systolic and diastolic blood pressure levels remained the same after treatment (121 ± 12/66 ± 7 before and 123 ± 11/67 ± 10 mm Hg after; p = 0.88 and 0.66, respectively). The study demonstrates that TNF-α inhibitors have no effect on blood pressure levels.

  18. Inhibitors of propagation of coagulation: factors V and X

    PubMed Central

    Toschi, Vincenzo; Lettino, Maddalena

    2011-01-01

    Cardiovascular diseases are still the most important cause of morbidity and mortality in western countries and antithrombotic treatment is nowadays widely used. Drugs able to reduce coagulation activation are the treatment of choice for a number of arterial and/or venous thromboembolic conditions. Some of the drugs currently used for this purpose, such as heparins (UFH or LMWH) and VKA, have limitations consisting of a narrow therapeutic window and an unpredictable response with the need of laboratory monitoring in order to assess their efficacy and safety. These drawbacks have stimulated an active research aimed to develop new drugs able to act on single factors involved in the coagulation network, with predictable response. Intense experimental and clinical work on new drugs has focused on synthetic agents, which could preferably be administered orally and at fixed doses. The most advanced clinical development with new anticoagulants has been achieved for those inhibiting FXa and some of them, like fondaparinux, are already currently used in clinical practice. Other agents, such as rivaroxaban, apixaban, otamixaban and edoxaban are under development and have already been studied or are currently under investigation in large scale phase III clinical trials for prevention and treatment of venous thromboembolism, atrial fibrillation and acute coronary syndromes. Some of them have proved to be more effective than conventional therapy. Data on some agents inhibiting FVa are still preliminary and some of these drugs have so far been considered only in patients with disseminated intravascular coagulation secondary to sepsis. PMID:21545479

  19. Predictive Factors for Efficacy of Dipeptidyl Peptidase-4 Inhibitors in Patients with Type 2 Diabetes Mellitus.

    PubMed

    Yagi, Shusuke; Aihara, Ken-Ichi; Akaike, Masashi; Fukuda, Daiju; Salim, Hotimah Masdan; Ishida, Masayoshi; Matsuura, Tomomi; Ise, Takayuki; Yamaguchi, Koji; Iwase, Takashi; Yamada, Hirotsugu; Soeki, Takeshi; Wakatsuki, Tetsuzo; Shimabukuro, Michio; Matsumoto, Toshio; Sata, Masataka

    2015-08-01

    Predictive factors for the efficacy of dipeptidyl peptidase-4 (DPP-4) inhibitors for lowering glycosylated hemoglobin (HbA1c) remain unclear in patients with type 2 diabetes mellitus. The aim of this study is therefore to clarify predictive factors of the efficacy of DPP-4 inhibitors for lowering HbA1c after 12 months of treatment. A total of 191 consecutive type 2 diabetic patients (male sex 55%, mean age, 68.3±35.8 years), who had been treated with DPP-4 inhibitors for 12 months, were enrolled in this study and evaluated retrospectively. After 12 months of DPP-4 inhibitor treatment, random blood glucose level, and HbA1c level, decreased from 167±63 to 151±49 mg/dL (P<0.01), and from 7.5%±1.3% to 6.9%±0.9% (P<0.01) respectively, without severe side effects. Multiple regression analysis showed that predictors of DPP-4 inhibitor treatment efficacy in lowering HbA1c level after 12 months were a decrease in HbA1c level after 3 months of treatment, a high baseline HbA1c level, a low baseline body mass index, and the absence of coronary artery disease. Most suitable candidates for treatment with DPP-4 inhibitors are diabetics who are not obese and do not have coronary artery disease. In addition, long-term efficacy of DPP-4 inhibitors can be predicted by decrement of HbA1c after 3 months of treatment.

  20. Factors Influencing Adherence in Cancer Patients Taking Oral Tyrosine Kinase Inhibitors: A Qualitative Study.

    PubMed

    Verbrugghe, Mathieu; Duprez, Veerle; Beeckman, Dimitri; Grypdonck, Mieke; Quaghebeur, Marijke; Verschueren, Caroline; Verhaeghe, Sofie; Van Hecke, Ann

    2016-01-01

    Nonadherence in cancer patients taking oral anticancer drugs is common. Reasons for nonadherence are still not really understood as influencing factors are often complex, dynamic, and interrelated. A qualitative study was conducted to gain insight into (non-)adherence behavior in patients taking oral tyrosine kinase inhibitors by exploring (1) processes and factors influencing (non-)adherence and (2) their interrelatedness. Semistructured interviews were held with 30 patients of different ages and with different types of cancer. A grounded theory approach was used. Three foci were found when dealing with oral tyrosine kinase inhibitors: (1) a focus on survival, (2) a focus on quality of life, and (3) a balance between survival and quality of life. The process of adherence was determined by a set of complex and interrelated influencing factors: treatment-related side effects, hope, anxiety, trust, and feedback mechanisms. This qualitative study gives insight into processes and factors influencing (non-)adherence behavior in patients taking oral tyrosine kinase inhibitors. The results of this study can help healthcare professionals understand why patients taking oral tyrosine kinase inhibitors do not always adhere to their therapy. Conditions should be created by which patients get maximum opportunity to establish a balance between survival and quality of life. An open climate and a trust-based relationship should be established in which patients feel comfortable to openly discuss their therapy and the difficulties they experience.

  1. IDO1 suppresses inhibitor development in hemophilia A treated with factor VIII

    PubMed Central

    Matino, Davide; Gargaro, Marco; Santagostino, Elena; Di Minno, Matteo N.D.; Castaman, Giancarlo; Morfini, Massimo; Rocino, Angiola; Mancuso, Maria E.; Di Minno, Giovanni; Coppola, Antonio; Talesa, Vincenzo N.; Volpi, Claudia; Vacca, Carmine; Orabona, Ciriana; Iannitti, Rossana; Mazzucconi, Maria G.; Santoro, Cristina; Tosti, Antonella; Chiappalupi, Sara; Sorci, Guglielmo; Tagariello, Giuseppe; Belvini, Donata; Radossi, Paolo; Landolfi, Raffaele; Fuchs, Dietmar; Boon, Louis; Pirro, Matteo; Marchesini, Emanuela; Grohmann, Ursula; Puccetti, Paolo; Iorio, Alfonso; Fallarino, Francesca

    2015-01-01

    The development of inhibitory antibodies to factor VIII (FVIII) is a major obstacle in using this clotting factor to treat individuals with hemophilia A. Patients with a congenital absence of FVIII do not develop central tolerance to FVIII, and therefore, any control of their FVIII-reactive lymphocytes relies upon peripheral tolerance mechanisms. Indoleamine 2,3-dioxygenase 1 (IDO1) is a key regulatory enzyme that supports Treg function and peripheral tolerance in adult life. Here, we investigated the association between IDO1 competence and inhibitor status by evaluating hemophilia A patients harboring F8-null mutations that were either inhibitor negative (n = 50) or positive (n = 50). We analyzed IDO1 induction, expression, and function for any relationship with inhibitor occurrence by multivariable logistic regression and determined that defective TLR9-mediated activation of IDO1 induction is associated with an inhibitor-positive status. Evaluation of experimental hemophilic mouse models with or without functional IDO1 revealed that tryptophan metabolites, which result from IDO1 activity, prevent generation of anti-FVIII antibodies. Moreover, treatment of hemophilic animals with a TLR9 agonist suppressed FVIII-specific B cells by a mechanism that involves IDO1-dependent induction of Tregs. Together, these findings indicate that strategies aimed at improving IDO1 function should be further explored for preventing or eradicating inhibitors to therapeutically administered FVIII protein. PMID:26426076

  2. Novel FXa Inhibitor Identification through Integration of Ligand- and Structure-Based Approaches.

    PubMed

    Lagos, Carlos F; Segovia, Gerardine F; Nuñez-Navarro, Nicolás; Faúndez, Mario A; Zacconi, Flavia C

    2017-09-22

    Factor Xa (FXa), a vitamin K-dependent serine protease plays a pivotal role in the coagulation cascade, one of the most interesting targets for the development of new anticoagulants. In the present work, we performed a virtual screening campaign based on ligand-based shape and electrostatic similarity search and protein-ligand docking to discover novel FXa-targeted scaffolds for further development of inhibitors. From an initial set of 260,000 compounds from the NCI Open database, 30 potential FXa inhibitors were identified and selected for in vitro biological evaluation. Compound 5 (NSC635393, 4-(3-methyl-4H-1,4-benzothiazin-2-yl)-2,4-dioxo-N-phenylbutanamide) displayed an IC50 value of 2.02 nM against human FXa. The identified compound may serve as starting point for the development of novel FXa inhibitors.

  3. Betrixaban compared with warfarin in patients with atrial fibrillation: results of a phase 2, randomized, dose-ranging study (Explore-Xa)

    PubMed Central

    Connolly, Stuart J.; Eikelboom, John; Dorian, Paul; Hohnloser, Stefan H.; Gretler, Daniel D.; Sinha, Uma; Ezekowitz, Michael D.

    2013-01-01

    Aims Patients with atrial fibrillation (AF) are at increased risk of stroke. Betrixaban is a novel oral factor Xa inhibitor administered once daily, mostly excreted unchanged in the bile and with low (17%) renal excretion. Methods and results Patients with AF and more than one risk factor for stroke were randomized to one of three blinded doses of betrixaban (40, 60, or 80 mg once daily) or unblinded warfarin, adjusted to an international normalized ratio of 2.0–3.0. The primary outcome was major or clinically relevant non-major bleeding. The mean follow-up was 147 days. Among 508 patients randomized, the mean CHADS2 score was 2.2; 87% of patients had previously received vitamin K antagonist therapy. The time in therapeutic range on warfarin was 63.4%. There were one, five, five, and seven patients with a primary outcome on betrixaban 40, 60, 80 mg daily, or warfarin, respectively. The rate of the primary outcome was lowest on betrixaban 40 mg (hazard ratio compared with warfarin = 0.14, exact stratified log-rank P-value 0.04, unadjusted for multiple testing). Rates of the primary outcome with betrixaban 60 or 80 mg were more similar to those of wafarin. Two ischaemic strokes occurred, one each on betrixaban 60 and 80 mg daily. There were two vascular deaths, one each on betrixaban 40 mg and warfarin. Betrixaban was associated with higher rates of diarrhoea than warfarin. Conclusion Betrixaban was well tolerated and had similar or lower rates of bleeding compared with well-controlled warfarin in patients with AF at risk for stroke. PMID:23487517

  4. Fast Rotation of a Subkilometer-sized Near-Earth Object 2011 XA3

    NASA Astrophysics Data System (ADS)

    Urakawa, Seitaro; Ohtsuka, Katsuhito; Abe, Shinsuke; Ito, Takashi; Nakamura, Tomoki

    2014-05-01

    We present light curve observations and their multiband photometry for near-Earth object (NEO) 2011 XA3. The light curve has shown a periodicity of 0.0304 ± 0.0003 days (= 43.8 ± 0.4 minutes). The fast rotation shows that 2011 XA3 is in a state of tension (i.e., a monolithic asteroid) and cannot be held together by self-gravitation. Moreover, the multiband photometric analysis indicates that the taxonomic class of 2011 XA3 is S-complex, or V-type. Its estimated effective diameter is 225 ± 97 m (S-complex) and 166 ± 63 m (V-type), respectively. Therefore, 2011 XA3 is a candidate for the second-largest, fast-rotating, monolithic asteroid. Moreover, the orbital parameters of 2011 XA3 are apparently similar to those of NEO (3200) Phaethon, but F/B-type. We computed the orbital evolutions of 2011 XA3 and Phaethon. However, the results of the computation and distinct taxonomy indicate that neither of the asteroids is of common origin.

  5. Fast rotation of a subkilometer-sized near-Earth object 2011 XA{sub 3}

    SciTech Connect

    Urakawa, Seitaro; Ohtsuka, Katsuhito; Abe, Shinsuke; Ito, Takashi; Nakamura, Tomoki

    2014-05-01

    We present light curve observations and their multiband photometry for near-Earth object (NEO) 2011 XA{sub 3}. The light curve has shown a periodicity of 0.0304 ± 0.0003 days (= 43.8 ± 0.4 minutes). The fast rotation shows that 2011 XA{sub 3} is in a state of tension (i.e., a monolithic asteroid) and cannot be held together by self-gravitation. Moreover, the multiband photometric analysis indicates that the taxonomic class of 2011 XA{sub 3} is S-complex, or V-type. Its estimated effective diameter is 225 ± 97 m (S-complex) and 166 ± 63 m (V-type), respectively. Therefore, 2011 XA{sub 3} is a candidate for the second-largest, fast-rotating, monolithic asteroid. Moreover, the orbital parameters of 2011 XA{sub 3} are apparently similar to those of NEO (3200) Phaethon, but F/B-type. We computed the orbital evolutions of 2011 XA{sub 3} and Phaethon. However, the results of the computation and distinct taxonomy indicate that neither of the asteroids is of common origin.

  6. A comparative approach expands the protein-protein interaction node of the immune receptor XA21 in wheat and rice

    PubMed Central

    Yang, Baoju; Ruan, Randy; Cantu, Dario; Wang, Xiaodong; Ji, Wanquan; Ronald, Pamela C; Dubcovsky, Jorge

    2016-01-01

    The rice (Oryza sativa) OsXA21 receptor kinase is a well-studied immune receptor that initiates a signal transduction pathway leading to resistance to Xanthomonas oryzae pv. oryzae. Two homologs of OsXA21 were identified in wheat (Triticum aestivum): TaXA21-like1 located in a syntenic region with OsXA21, and TaXA21-like2 located in a non-syntenic region. Proteins encoded by these two wheat genes interact with four wheat orthologs of known OsXA21 interactors. In this study, we screened a wheat yeast-two-hybrid (Y2H) library using the cytosolic portion of TaXA21-like1 as bait to identify additional interactors. Using full-length T. aestivum and T. monococcum proteins and Y2H assays we identified three novel TaXA21-like1 interactors (TaARG, TaPR2, TmSKL1) plus one previously known in rice (TaSGT1). An additional full-length wheat protein (TaCIPK14) interacted with TaXA21-like2 and OsXA21 but not with TaXA21-like1. The interactions of TaXA21-like1 with TmSKL1 and TaSGT1 were also observed in rice protoplasts using bimolecular fluorescence complementation (BiFC) assays. We then cloned the rice homologs of the novel wheat interactors and confirmed that they all interact with OsXA21. This last result suggests that inter-specific comparative interactome analyses can be used not only to transfer known interactions from rice to wheat, but also to identify novel interactions in rice. PMID:23957671

  7. Concomitant disseminated histoplasmosis and disseminated tuberculosis after tumor necrosis factor inhibitor treatment: a case report.

    PubMed

    Muñoz-Oca, Juan E; Villarreal Morales, Martha L; Nieves-Rodriguez, Aracelis; Martínez-Bonilla, Lemuel

    2017-01-13

    Tumor necrosis factor antagonist inhibitors have transformed the approach to patients with severe autoimmune conditions, such as rheumatoid arthritis. Although the therapy can be highly effective, TNF-α inhibitors are associated with an increased risk of opportunistic infections. Here, we report a case of concomitant disseminated histoplasmosis and tuberculosis in a 65-year-old female with rheumatoid arthritis treated with TNF-α inhibitor. Both conditions can be found in disseminated form in immunosuppressed hosts, but co-infection is rare with only a few cases having been reported, to our knowledge, all in HIV patients. This case posed a considerable challenge for diagnosis and treatment due to the unusual disseminated co-infection, the overlapping symptoms, and the interactions between medications.

  8. Nonneutralizing antibodies against factor VIII and risk of inhibitor development in severe hemophilia A.

    PubMed

    Cannavò, Antonino; Valsecchi, Carla; Garagiola, Isabella; Palla, Roberta; Mannucci, Pier Mannuccio; Rosendaal, Frits R; Peyvandi, Flora

    2017-03-09

    The development of anti-factor VIII (FVIII) neutralizing antibodies (inhibitors) is the major complication in hemophilia A. Nonneutralizing antibodies (NNAs) have been detected in hemophilia patients and also in unaffected individuals. The aim of this study was to assess the prevalence of NNAs and to evaluate whether their presence is associated with the development of inhibitors in a cohort of previously untreated or minimally treated patients with hemophilia A; plasma samples of 237 patients with severe hemophilia A enrolled in the SIPPET trial were collected before any exposure to FVIII concentrates and analyzed for the presence of anti-FVIII NNAs. Patients were observed for the development of neutralizing antibodies. NNAs were found in 18 (7.6%) of 237 patients at screening, and there was a clear age gradient. Of those with NNAs, 7 patients subsequently developed an inhibitor for a cumulative incidence of 45.4% (95% confidence interval [CI], 19.5% to 71.3%); among the 219 patients without NNAs, 64 (29%) developed an inhibitor (cumulative incidence, 34.0%; 95% CI, 27.1%-40.9%). In Cox regression analyses, patients with NNAs at screening had an 83% higher incidence of inhibitor development than patients without NNAs (hazard ratio [HR], 1.83; 95% CI, 0.84-3.99). For high-titer inhibitors, the incidence rate had an almost threefold increase (HR, 2.74; 95% CI, 1.23-6.12). These associations did not materially change after adjustment. The presence of anti-FVIII NNAs in patients with severe hemophilia A who were not previously exposed to FVIII concentrates is associated with an increased incidence of inhibitors. © 2017 by The American Society of Hematology.

  9. Factors associated with virological rebound in HIV-infected patients receiving protease inhibitor monotherapy.

    PubMed

    Stöhr, Wolfgang; Dunn, David T; Arenas-Pinto, Alejandro; Orkin, Chloe; Clarke, Amanda; Williams, Ian; Johnson, Margaret; Beeching, Nicholas J; Wilkins, Edmund; Sanders, Karen; Paton, Nicholas I

    2016-11-13

    The Protease Inhibitor Monotherapy Versus Ongoing Triple Therapy (PIVOT) trial found that protease inhibitor monotherapy as a simplification strategy is well tolerated in terms of drug resistance but less effective than combination therapy in suppressing HIV viral load. We sought to identify factors associated with the risk of viral load rebound in this trial. PIVOT was a randomized controlled trial in HIV-positive adults with suppressed viral load for at least 24 weeks on combination therapy comparing a strategy of physician-selected ritonavir-boosted protease inhibitor monotherapy versus ongoing triple therapy. In participants receiving monotherapy, we analysed time to confirmed viral load rebound and its predictors using flexible parametric survival models. Of 290 participants initiating protease inhibitor monotherapy (80% darunavir, 14% lopinavir, and 6% other), 93 developed viral load rebound on monotherapy. The risk of viral load rebound peaked at 9 months after starting monotherapy and then declined to approximately 5 per 100 person-years from 18 months onwards. Independent predictors of viral load rebound were duration of viral load suppression before starting monotherapy (hazard ratio 0.81 per additional year <50 copies/ml; P < 0.001), CD4 cell count (hazard ratio 0.73 per additional 100 cells/μl for CD4 nadir; P = 0.008); ethnicity (hazard ratio 1.87 for nonwhite versus white, P = 0.025) but not the protease inhibitor agent used (P = 0.27). Patients whose viral load was analysed with the Roche TaqMan-2 assay had a 1.87-fold risk for viral load rebound compared with Abbott RealTime assay (P = 0.012). A number of factors can identify patients at low risk of rebound with protease inhibitor monotherapy, and this may help to better target those who may benefit from this management strategy.

  10. Risk Factors for Inhibitor Formation in Hemophilia: A Prevalent Case-Control Study

    PubMed Central

    Ragni, Margaret V.; Ojeifo, Oluseyi; Feng, Jinong; Yan, Jin; Hill, Kathleen A.; Sommer, Steve S.; Trucco, Massimo N.; Brambilla, Donald J.

    2009-01-01

    Background Inhibitor formation is a major complication of hemophilia treatment. Aim In a prevalent case-control study, we evaluated blood product exposure, genotype, and HLA type on hemophilia A inhibitor formation. Methods Product exposure was extracted from medical records. Genotype was determined on stored DNA samples by detection of virtually all mutations-SSCP (DOVAM-S) and subcycling PCR. HLA typing was performed by PCR amplification and exonuclease-released fluorescence. Results Cases experienced higher intensity factor, 455 vs. 200 U per exposure, p<0.005, more frequent central nervous system (CNS) bleeding, 7 of 20 (35.0%) vs. 1 of 57 (1.7%), p=0.001, and more commonly from inhibitor families, 7 of 20 (35.0%) vs. 0 of 57 (0%), p<0.001, and African-American, 12 of 63 (19.0%) vs. 6 of 117 (5.1%), p=0.015. Among the latter, CNS bleeding was more commonly the initial bleed, 60% vs. 0%, p<0.001, and survival was shorter, 14 vs. 38 yr, p=0.025. Inhibitor formation was uncommon in those with missense mutations, 2 of 65 (3.1%) vs. 31 of 119 (26.0%), p=0.008, and unrelated to factor VIII immunogenic epitope, p=0.388, or HLA type, p>0.100. Genotype was not associated with race. Time to immune tolerance was shorter for titers < 120 vs. ≥ 120 BU/ml, 6 vs. 16 months, p<0.01, but unaffected by tolerizing dose regimen, p>0.50. Conclusions Inhibitor formation is associated with high intensity product exposure, CNS bleeding, African-American race, and low frequency of missense mutations. The ideal time to initiate prophylaxis to reduce CNS bleeding and inhibitor formation will require prospective studies. PMID:19563499

  11. Pharmacophore selection and redesign of non-nucleotide inhibitors of anthrax edema factor.

    PubMed

    Schein, Catherine H; Chen, Deliang; Ma, Lili; Kanalas, John J; Gao, Jian; Jimenez, Maria Estrella; Sower, Laurie E; Walter, Mary A; Gilbertson, Scott R; Peterson, Johnny W

    2012-11-08

    Antibiotic treatment may fail to protect individuals, if not started early enough, after infection with Bacillus anthracis, due to the continuing activity of toxins that the bacterium produces. Stable and easily stored inhibitors of the edema factor toxin (EF), an adenylyl cyclase, could save lives in the event of an outbreak, due to natural causes or a bioweapon attack. The toxin's basic activity is to convert ATP to cAMP, and it is thus in principle a simple phosphatase, which means that many mammalian enzymes, including intracellular adenylcyclases, may have a similar activity. While nucleotide based inhibitors, similar to its natural substrate, ATP, were identified early, these compounds had low activity and specificity for EF. We used a combined structural and computational approach to choose small organic molecules in large, web-based compound libraries that would, based on docking scores, bind to residues within the substrate binding pocket of EF. A family of fluorenone-based inhibitors was identified that inhibited the release of cAMP from cells treated with EF. The lead inhibitor was also shown to inhibit the diarrhea caused by enterotoxigenic E. coli (ETEC) in a murine model, perhaps by serving as a quorum sensor. These inhibitors are now being tested for their ability to inhibit Anthrax infection in animal models and may have use against other pathogens that produce toxins similar to EF, such as Bordetella pertussis or Vibrio cholera.

  12. Accuracy and consistency of anti-Xa activity measurement for determination of rivaroxaban plasma levels.

    PubMed

    Studt, J-D; Alberio, L; Angelillo-Scherrer, A; Asmis, L M; Fontana, P; Korte, W; Mendez, A; Schmid, P; Stricker, H; Tsakiris, D A; Wuillemin, W A; Nagler, M

    2017-08-01

    Essentials Accurate determination of anticoagulant plasma concentration is important in clinical practice. We studied the accuracy and consistency of anti-Xa assays for rivaroxaban in a multicentre study. In a range between 50 and 200 μg L(-1) , anti-Xa activity correlated well with plasma concentrations. The clinical value might be limited by overestimation and intra- and inter-individual variation. Background Determining the plasma level of direct oral anticoagulants reliably is important in the work-up of complex clinical situations. Objectives To study the accuracy and consistency of anti-Xa assays for rivaroxaban plasma concentration in a prospective, multicenter evaluation study employing different reagents and analytical platforms. Methods Rivaroxaban 20 mg was administered once daily to 20 healthy volunteers and blood samples were taken at peak and trough levels (clinicaltrials.gov NCT01710267). Anti-Xa activity was determined in 10 major laboratories using different reagents and analyzers; corresponding rivaroxaban plasma concentrations were measured by high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS). Findings Overall Pearson's correlation coefficient of anti-Xa levels and HPLC-MS results was 0.99 for Biophen(®) Heparin (95% CI, 0.99, 0.99), Biophen(®) DiXaI (95% CI, 0.99, 0.99) and STA(®) anti-Xa liquid (95% CI, 0.99, 1.00). Correlation was lower in rivaroxaban concentrations below 50 μg L(-1) and above 200 μg L(-1) . The overall bias of the Bland-Altman difference plot was 14.7 μg L(-1) for Biophen Heparin, 17.9 μg L(-1) for Biophen DiXal and 19.0 μg L(-1) for STA anti-Xa liquid. Agreement between laboratories was high at peak level but limited at trough level. Conclusions Anti-Xa activity correlated well with rivaroxaban plasma concentrations, especially in a range between 50 and 200 μg L(-1) . However, anti-Xa assays systematically overestimated rivaroxaban concentration as compared with HPLC

  13. Unfractionated heparin dose requirements targeting intermediate intensity antifactor Xa concentration during pregnancy.

    PubMed

    Clark, Nathan P; Delate, Thomas; Cleary, Steven J; Witt, Daniel M

    2010-04-01

    To describe unfractionated heparin (UFH) dosing requirements and monitoring parameters to achieve target antifactor Xa concentrations in pregnant women receiving intermediate-dose UFH therapy. Retrospective cohort analysis. Centralized anticoagulation service in an integrated health care delivery system. Twenty-five pregnant women (who had 27 pregnancies) who received intermediate-dose UFH between January 1, 1998, and March 31, 2005. Demographic and clinical data were retrieved by using an integrated electronic medical, pharmacy, and laboratory records system. The primary outcome was the required UFH dose at the time of first target antifactor Xa level achieved. Antifactor Xa levels were measured at the mid-dosing interval for UFH. An antifactor Xa assay concentration of 0.1-0.3 unit/ml was used as the target range for intermediate-dose UFH for antithrombotic management of pregnant women as described in the American College of Chest Physicians clinical practice guidelines. The mean UFH dose required to achieve this target antifactor Xa range was 236.9 units/kg/day. The UFH doses required to achieve target antifactor Xa levels correlated significantly with patient weight (r = 0.682, p<0.001). The final UFH dose/kg required at the end of pregnancy was similar to that at the first target level (p>0.05) when adjusted for weight. However, the total daily amount of UFH did increase by the end of pregnancy (21,889 vs 19,320 units, p=0.02). There was an average of 7.9 antifactor Xa determinations and 3.1 dosage modifications during the mean of 19 weeks of antenatal UFH therapy. Most (62%) antifactor Xa levels were within the target range. No thromboembolic events, heparin-induced thrombocytopenia, or osteopenic fracture occurred. There was one hemorrhagic complication that resolved with temporary withdrawal of UFH. Pregnant women required a mean UFH dose of 236.9 units/kg/day to achieve the targeted antifactor Xa level of 0.1-0.3 unit/ml. The required UFH doses correlated

  14. Inhibition of the activation of Hageman factor (factor XII) by peripheral blood cells.

    PubMed Central

    Ratnoff, O D; Emanuelson, M M; Ziats, N P

    1987-01-01

    Suspensions of peripheral blood mononuclear cells (PBMC), monocytes, T or B lymphocytes, platelets or granulocytes, and cell-depleted supernatant fluids of these suspensions inhibited activation of Hageman factor (HF, Factor XII) by ellagic acid, a property not shared by erythrocytes. PBMC also inhibited HF activation by glass or sulfatides. Contaminating platelets may have contributed to inhibition by PBMC. Elaboration of agents inhibiting HF activation required metabolically active cells. The inhibitor(s) in PBMC supernates were not identified with known agents, but had properties of a nonenzymatic protein. PBMC supernates did not contain fibrinogen, nor alter the thrombin, prothrombin, or partial thromboplastin times of normal plasma, amidolysis by activated plasma thromboplastin antecedent (Factor XIa) or activated Stuart factor (Factor Xa) or esterolysis by C1 (C1 esterase); they inhibited plasmin minimally. These experiments suggest that peripheral blood cells may impede intravascular coagulation. Whether this property helps maintain the fluidity of blood is unclear. PMID:3498741

  15. ADAMTS13 Endopeptidase Protects against Vascular Endothelial Growth Factor Inhibitor-Induced Thrombotic Microangiopathy.

    PubMed

    Erpenbeck, Luise; Demers, Melanie; Zsengellér, Zsuzsanna K; Gallant, Maureen; Cifuni, Stephen M; Stillman, Isaac E; Karumanchi, S Ananth; Wagner, Denisa D

    2016-01-01

    Thrombotic microangiopathy (TMA) is a life-threatening condition that affects some, but not all, recipients of vascular endothelial growth factor (VEGF) inhibitors given as part of chemotherapy. TMA is also a complication of preeclampsia, a disease characterized by excess production of the VEGF-scavenging soluble VEGF receptor 1 (soluble fms-like tyrosine kinase 1; sFlt-1). Risk factors for VEGF inhibitor-related TMA remain unknown. We hypothesized that deficiency of the VWF-cleaving ADAMTS13 endopeptidase contributes to the development of VEGF inhibitor-related TMA. ADAMTS13(-/-) mice overexpressing sFlt-1 presented all hallmarks of TMA, including thrombocytopenia, schistocytosis, anemia, and VWF-positive microthrombi in multiple organs. Similar to VEGF inhibitor-related TMA in humans, these mice exhibited severely impaired kidney function and hypertension. In contrast, wild-type mice overexpressing sFlt-1 developed modest hypertension but no other features of TMA. Recombinant ADAMTS13 therapy ameliorated all symptoms of TMA in ADAMTS13(-/-) mice overexpressing sFlt-1 and normalized BP in wild-type mice. ADAMTS13 activity may thus be a critical determinant for the development of TMA secondary to VEGF inhibition. Administration of recombinant ADAMTS13 may serve as a therapeutic approach to treat or prevent thrombotic complications of VEGF inhibition.

  16. Prolonged Activated Partial Thromboplastin Time: Difficulties in Discriminating Coexistent Factor VIII Inhibitor and Lupus Anticoagulant.

    PubMed

    Ames, Paul R J; Graf, Maria; Archer, Jeremy; Scarpato, Nicola; Iannaccone, Luigi

    2015-03-01

    To review the diagnostic difficulties of a prolonged activated partial thromboplastin time (aPTT) when 2 inhibitors with opposite clinical presentations coexist, we searched MEDLINE from January 1970 to November 2013 using acquired, factor VIII (FVIII), factor IX, hemophilia A and B, inhibitor, lupus anticoagulant (LA), antiphospholipid, anticardiolipin, anti-β2-glycoprotein I, antibodies, syndrome, bleeding, and thrombosis. We identified 13 articles for a total of 15 cases of possible coexistence of FVIII inhibitor and LA. The presenting clinical manifestation was thrombosis in 6 cases and bleeding in 9 cases. Activated partial thromboplastin time was the presenting laboratory abnormality in all cases, and first-line investigations suggested the coexistence of LA and acquired FVIII inhibitor. None of the articles addressed the diagnostic accuracy of the screening tests by performing "second line" assays. We reviewed the diagnostic pitfalls of the cases under study and provide some guidance for alternative tests when facing a prolonged aPTT that may have a double meaning.

  17. Yeast-hybrid based high-throughput assay for identification of anthrax lethal factor inhibitors.

    PubMed

    Kim, Joungmok; Park, Hae-Chul; Gedi, Vinayakumar; Park, Hye-Yeon; Roberts, Arthur G; Atkins, William M; Yoon, Moon-Young

    2011-01-07

    Inhibitors of anthrax lethal factor (LF) are currently being sought as effective therapeutics for the treatment of anthrax. Here we report a novel screening approach for inhibitors of LF, a yeast-hybrid-based assay system in which the expression of reporter genes from a Gal4 promoter is repressed by LF proteolytic activity. Yeast cells were co-transformed with LF and a chimeric transcription factor that contains an LF substrate sequence inserted between the DNA-binding and activation domains of Gal4. In the resulting yeast cells, LF cleaves the substrate, thus inactivating the chimeric Gal4 and resulting in lack of expression of reporter genes. Compounds that inhibit LF cleavage of its substrate are identified by changes in reporter gene activity. Relative to in vitro screens for inhibitors of LF proteolytic activity, this screen has the advantage of excluding compounds that are toxic or non-permeable to eukaryotic cells. Additionally, the screen has the advantage of being fast, easy and cheap because exogenous LF and substrate are not needed. An initial chemical library screen with this system has identified four candidate inhibitors which were confirmed to inhibit LF protease activity in an in vitro assay. Furthermore, FBS-00831, one of the compounds identified, protects Raw 264.7 macrophages from anthrax lethal toxin and the possible binding site on LF was also evaluated by molecular docking.

  18. Vascular endothelial growth factor inhibitors: investigational therapies for the treatment of psoriasis

    PubMed Central

    Weidemann, Anja K; Crawshaw, Ania A; Byrne, Emily; Young, Helen S

    2013-01-01

    Psoriasis is a common inflammatory autoimmune condition in which environmental factors and genetic predisposition contribute to the development of disease in susceptible individuals. Angiogenesis is known to be a key pathogenic feature of psoriasis. Local and systemic elevation of vascular endothelial growth factor (VEGF)-A has been demonstrated in the skin and plasma of patients with psoriasis and is known to correlate with improvement following some traditional psoriasis treatments. A number of VEGF inhibitors are licensed for the treatment of malignancies and eye disease and isolated case reports suggest that some individuals with psoriasis may improve when exposed to these agents. The small number of cases and lack of unified reporting measures makes it difficult to draw generalizations and underline the heterogeneity of psoriasis as a disease entity. Though not yet licensed for the treatment of psoriasis in humans, experimental data supports the potential of VEGF inhibitors to influence relevant aspects of human cell biology (such as endothelial cell differentiation) and to improve animal models of skin disease. Given the multi-factorial nature of psoriasis it is unlikely that VEGF inhibitors will be effective in all patients, however they have the potential to be a valuable addition to the therapeutic arsenal in selected cases. Current VEGF inhibitors in clinical use are associated with a number of potentially serious side effects including hypertension, left ventricular dysfunction, and gastrointestinal perforation. Such risks require careful consideration in psoriasis populations particularly in light of growing concerns linking psoriasis to increased cardiovascular risk. PMID:24101875

  19. Effect of nadroparin on anti-Xa activity during nocturnal hemodialysis

    PubMed Central

    Buitenwerf, Edward; Risselada, Arne J.; van Roon, Eric N.; Veeger, Nic J.G.M.; Hemmelder, Marc H.

    2015-01-01

    Background Nadroparin is used during hemodialysis to prevent clotting of the extra corporeal system. During nocturnal hemodialysis patients receive an increased dosage of nadroparin compared to conventional hemodialysis. We tested whether the prescribed dosage regimen of nadroparin, according to Dutch guidelines, causes accumulation of nadroparin. Methods Anti-Xa levels were used as an indicator of nadroparin accumulation. Anti-Xa was measured photometrically in 13 patients undergoing nocturnal hemodialysis for 4 nights a week. Nadroparin was administered according to Dutch dosage guidelines. We assessed anti-Xa levels at 4 time points during 1 dialysis week: before the start of the first dialysis session of the week (baseline), prior to (T1) and after the last dialysis session of the week (T2) and before the first dialysis of the following week (T3). Results Patients received 71–95 IU/kg at the start of dialysis and another 50% of the initial dosage after 4 h with a total cumulative dosage of 128 ± 24 IU/kg. Anti-Xa levels increased from 0.017 at baseline to 0.019 at T1 (p = 0.03). Anti-Xa levels were 0.419 ± 0.252 IU/ml at T2 (p < 0.001 vs baseline and T1), whereas anti-Xa levels were not changed at T3 compared to baseline. Conclusion Dosing of nadroparin according to Dutch guidelines in patients on nocturnal hemodialysis does not lead to accumulation of nadroparin. We therefore consider the Dutch dosage guidelines for nadroparin an effective and safe strategy. General significance This article is the first to present data on anti-Xa activity during nocturnal hemodialysis which is a widely used and potentially dangerous therapy. PMID:26672512

  20. KRAS Testing and Epidermal Growth Factor Receptor Inhibitor Treatment for Colorectal Cancer in Community Settings

    PubMed Central

    Webster, Jennifer; Kauffman, Tia L.; Feigelson, Heather Spencer; Pawloski, Pamala A.; Onitilo, Adedayo A.; Potosky, Arnold L.; Cross, Deanna; Meier, Paul R.; Mirabedi, Anousheh S.; Delate, Thomas; Daida, Yihe; Williams, Andrew E.; Alexander, Gwen L.; McCarty, Catherine A.; Honda, Stacey; Kushi, Lawrence H.; Goddard, Katrina A.B.

    2013-01-01

    Background In metastatic colorectal cancer (mCRC), mutations in the KRAS gene predict poor response to epidermal growth factor receptor (EGFR) inhibitors. Clinical treatment guidelines now recommend KRAS testing if EGFR inhibitors are considered. Our study investigates the clinical uptake and utilization of KRAS testing. Methods We included 1,188 patients with mCRC diagnosed from 2004 to 2009, from seven integrated health care delivery systems with a combined membership of 5.5 million. We used electronic medical records and targeted manual chart review to capture the complexity and breadth of real-world clinical oncology care. Results Overall, 428 patients (36%) received KRAS testing during their clinical care, and 266 (22%) were treated with EGFR inhibitors. Age at diagnosis (p=0.0034), comorbid conditions (p=0.0316), and survival time from diagnosis (p<0.0001) influence KRAS testing and EGFR inhibitor prescribing. The proportion who received KRAS testing increased from 7% to 97% for those treated in 2006 and 2010, respectively, and 83% of all treated patients had a KRAS wild type genotype. Most patients with a KRAS mutation (86%) were not treated with EGFR inhibitors. The interval between mCRC diagnosis and receipt of KRAS testing decreased from 26 months (2006) to 10 months (2009). Conclusions These findings demonstrate rapid uptake and incorporation of this predictive biomarker into clinical oncology care. Impact In this delivery setting, KRAS testing is widely used to guide treatment decisions with EGFR inhibitors in patients with mCRC. An important future research goal is to evaluate utilization of KRAS testing in other delivery settings in the US. PMID:23155138

  1. Acquired Factor V Inhibitors in a Patient with End-stage Renal Disease

    PubMed Central

    Kitazawa, Atsushi; Misawa, Hideo; Nagahori, Katsuhiro; Koda, Ryo; Yoshino, Atsunori; Kawamoto, Shinya; Takeda, Tetsuro

    2016-01-01

    We report a case of acquired factor V inhibitors (AFVIs) in a patient with end-stage renal disease receiving warfarin therapy for atrial fibrillation. A 72-year-old Japanese man was admitted to our hospital complaining of tarry stools and abdominal pain. The laboratory findings revealed eosinophilia (52.1%), prolonged activated partial thromboplastin time (APTT) (98 s), PT (84 s), a factor V (FV) activity of <3%, and an FV inhibitor level of 6 Bethesda units/mL. After administration of prednisolone was started, his coagulation findings improved. However, his renal failure progressed, and he ultimately required chronic hemodialysis. This is the first case of AFVIs in a patient starting hemodialysis for end-stage renal disease. PMID:27904118

  2. Acquired Factor V Inhibitors in a Patient with End-stage Renal Disease.

    PubMed

    Kitazawa, Atsushi; Misawa, Hideo; Nagahori, Katsuhiro; Koda, Ryo; Yoshino, Atsunori; Kawamoto, Shinya; Takeda, Tetsuro

    We report a case of acquired factor V inhibitors (AFVIs) in a patient with end-stage renal disease receiving warfarin therapy for atrial fibrillation. A 72-year-old Japanese man was admitted to our hospital complaining of tarry stools and abdominal pain. The laboratory findings revealed eosinophilia (52.1%), prolonged activated partial thromboplastin time (APTT) (98 s), PT (84 s), a factor V (FV) activity of <3%, and an FV inhibitor level of 6 Bethesda units/mL. After administration of prednisolone was started, his coagulation findings improved. However, his renal failure progressed, and he ultimately required chronic hemodialysis. This is the first case of AFVIs in a patient starting hemodialysis for end-stage renal disease.

  3. Identification of inhibitors for vascular endothelial growth factor receptor by using dynamic combinatorial chemistry.

    PubMed

    Yang, Zhao; Fang, Zheng; He, Wei; Wang, Zhixiang; Gan, Haifeng; Tian, Qitao; Guo, Kai

    2016-04-01

    The novel analysis method consisting of size-exclusion chromatography (SEC) and HRMS analysis was firstly applied in the discovery of potential inhibitors towards cancer drug targets. With vascular endothelial growth factor receptor (VEGFR-2) as a target, dynamic combinatorial libraries (DCLs) were prepared by reacting aldehydes with amines. Four sensitive binders targeted VEGFR-2 were directly isolated from the library. Antitumor activity test in vitro and inhibition experiments toward angiogenesis were also carried out.

  4. JAK3 inhibitor VI is a mutant specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation T790M

    PubMed Central

    Nishiya, Naoyuki; Sakamoto, Yasumitsu; Oku, Yusuke; Nonaka, Takamasa; Uehara, Yoshimasa

    2015-01-01

    AIM: To identify non-quinazoline kinase inhibitors effective against drug resistant mutants of epidermal growth factor receptor (EGFR). METHODS: A kinase inhibitor library was subjected to screening for specific inhibition pertaining to the in vitro kinase activation of EGFR with the gatekeeper mutation T790M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in non-small cell lung cancers (NSCLCs). This inhibitory effect was confirmed by measuring autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells, an NSCLC cell line harboring the gatekeeper mutation. The effects of a candidate compound, Janus kinase 3 (JAK3) inhibitor VI, on cell proliferation were evaluated using the MTT assay and were compared between T790M-positive and -negative lung cancer cell lines. JAK3 inhibitor VI was modeled into the ATP-binding pocket of EGFR T790M/L858R. Potential physical interactions between the compound and kinase domains of wild-type (WT) or mutant EGFRs or JAK3 were estimated by calculating binding energy. The gatekeeper residues of EGFRs and JAKs were aligned to discuss the similarities among EGFR T790M and JAKs. RESULTS: We found that JAK3 inhibitor VI, a known inhibitor for JAK3 tyrosine kinase, selectively inhibits EGFR T790M/L858R, but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor VI also specifically reduced autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells upon EGF stimulation, but did not show the inhibitory effect on WT EGFR in A431 cells. Furthermore, JAK3 inhibitor VI suppressed the proliferation of NCI-H1975 cells, but showed limited inhibitory effects on the WT EGFR-expressing cell lines A431 and A549. A docking simulation between JAK3 inhibitor VI and the ATP-binding pocket of EGFR T790M/L858R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor VI to EGFR T790M/L858R was more stable than its binding energy to the WT EGFR. Amino acid

  5. JAK3 inhibitor VI is a mutant specific inhibitor for epidermal growth factor receptor with the gatekeeper mutation T790M.

    PubMed

    Nishiya, Naoyuki; Sakamoto, Yasumitsu; Oku, Yusuke; Nonaka, Takamasa; Uehara, Yoshimasa

    2015-11-26

    To identify non-quinazoline kinase inhibitors effective against drug resistant mutants of epidermal growth factor receptor (EGFR). A kinase inhibitor library was subjected to screening for specific inhibition pertaining to the in vitro kinase activation of EGFR with the gatekeeper mutation T790M, which is resistant to small molecular weight tyrosine kinase inhibitors (TKIs) for EGFR in non-small cell lung cancers (NSCLCs). This inhibitory effect was confirmed by measuring autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells, an NSCLC cell line harboring the gatekeeper mutation. The effects of a candidate compound, Janus kinase 3 (JAK3) inhibitor VI, on cell proliferation were evaluated using the MTT assay and were compared between T790M-positive and -negative lung cancer cell lines. JAK3 inhibitor VI was modeled into the ATP-binding pocket of EGFR T790M/L858R. Potential physical interactions between the compound and kinase domains of wild-type (WT) or mutant EGFRs or JAK3 were estimated by calculating binding energy. The gatekeeper residues of EGFRs and JAKs were aligned to discuss the similarities among EGFR T790M and JAKs. We found that JAK3 inhibitor VI, a known inhibitor for JAK3 tyrosine kinase, selectively inhibits EGFR T790M/L858R, but has weaker inhibitory effects on the WT EGFR in vitro. JAK3 inhibitor VI also specifically reduced autophosphorylation of EGFR T790M/L858R in NCI-H1975 cells upon EGF stimulation, but did not show the inhibitory effect on WT EGFR in A431 cells. Furthermore, JAK3 inhibitor VI suppressed the proliferation of NCI-H1975 cells, but showed limited inhibitory effects on the WT EGFR-expressing cell lines A431 and A549. A docking simulation between JAK3 inhibitor VI and the ATP-binding pocket of EGFR T790M/L858R predicted a potential binding status with hydrogen bonds. Estimated binding energy of JAK3 inhibitor VI to EGFR T790M/L858R was more stable than its binding energy to the WT EGFR. Amino acid sequence alignments revealed

  6. Molecular modeling studies of [6,6,5] Tricyclic Fused Oxazolidinones as FXa inhibitors using 3D-QSAR, Topomer CoMFA, molecular docking and molecular dynamics simulations.

    PubMed

    Xu, Cheng; Ren, Yujie

    2015-10-15

    Coagulation factor Xa (Factor Xa, FXa) is a particularly promising target for novel anticoagulant therapy. The first oral factor Xa inhibitor has been approved in the EU and Canada in 2008. In this work, 38 [6,6,5] Tricyclic Fused Oxazolidinones were studied using a combination of molecular modeling techniques including three-dimensional quantitative structure-activity relationship (3D-QSAR), molecular docking, molecular dynamics and Topomer CoMFA (comparative molecular field analysis) were used to build 3D-QSAR models. The results show that the best CoMFA model has q(2)=0.511 and r(2)=0.984, the best CoMSIA (comparative molecular similarity indices analysis) model has q(2)=0.700 and r(2)=0.993 and the Topomer CoMFA analysis has q(2)=0.377 and r(2)=0.886. The results indicated the steric, hydrophobic, H-acceptor and electrostatic fields play key roles in models. Molecular docking and molecular dynamics explored the binding relationship of the ligand and the receptor protein. Copyright © 2015 Elsevier Ltd. All rights reserved.

  7. 3D/3D registration of coronary CTA and biplane XA reconstructions for improved image guidance.

    PubMed

    Dibildox, Gerardo; Baka, Nora; Punt, Mark; Aben, Jean-Paul; Schultz, Carl; Niessen, Wiro; van Walsum, Theo

    2014-09-01

    The authors aim to improve image guidance during percutaneous coronary interventions of chronic total occlusions (CTO) by providing information obtained from computed tomography angiography (CTA) to the cardiac interventionist. To this end, the authors investigate a method to register a 3D CTA model to biplane reconstructions. The authors developed a method for registering preoperative coronary CTA with intraoperative biplane x-ray angiography (XA) images via 3D models of the coronary arteries. The models are extracted from the CTA and biplane XA images, and are temporally aligned based on CTA reconstruction phase and XA ECG signals. Rigid spatial alignment is achieved with a robust probabilistic point set registration approach using Gaussian mixture models (GMMs). This approach is extended by including orientation in the Gaussian mixtures and by weighting bifurcation points. The method is evaluated on retrospectively acquired coronary CTA datasets of 23 CTO patients for which biplane XA images are available. The Gaussian mixture model approach achieved a median registration accuracy of 1.7 mm. The extended GMM approach including orientation was not significantly different (P>0.1) but did improve robustness with regards to the initialization of the 3D models. The authors demonstrated that the GMM approach can effectively be applied to register CTA to biplane XA images for the purpose of improving image guidance in percutaneous coronary interventions.

  8. 3D/3D registration of coronary CTA and biplane XA reconstructions for improved image guidance

    SciTech Connect

    Dibildox, Gerardo Baka, Nora; Walsum, Theo van; Punt, Mark; Aben, Jean-Paul; Schultz, Carl; Niessen, Wiro

    2014-09-15

    Purpose: The authors aim to improve image guidance during percutaneous coronary interventions of chronic total occlusions (CTO) by providing information obtained from computed tomography angiography (CTA) to the cardiac interventionist. To this end, the authors investigate a method to register a 3D CTA model to biplane reconstructions. Methods: The authors developed a method for registering preoperative coronary CTA with intraoperative biplane x-ray angiography (XA) images via 3D models of the coronary arteries. The models are extracted from the CTA and biplane XA images, and are temporally aligned based on CTA reconstruction phase and XA ECG signals. Rigid spatial alignment is achieved with a robust probabilistic point set registration approach using Gaussian mixture models (GMMs). This approach is extended by including orientation in the Gaussian mixtures and by weighting bifurcation points. The method is evaluated on retrospectively acquired coronary CTA datasets of 23 CTO patients for which biplane XA images are available. Results: The Gaussian mixture model approach achieved a median registration accuracy of 1.7 mm. The extended GMM approach including orientation was not significantly different (P > 0.1) but did improve robustness with regards to the initialization of the 3D models. Conclusions: The authors demonstrated that the GMM approach can effectively be applied to register CTA to biplane XA images for the purpose of improving image guidance in percutaneous coronary interventions.

  9. A molecular modeling study of inhibitors of nuclear factor kappa-B (p50)--DNA binding.

    PubMed

    Pande, Vineet; Sharma, Rakesh K; Inoue, Jun-Ichiro; Otsuka, Masami; Ramos, Maria J

    2003-12-01

    Nuclear Factor-kappa B (NF-kappaB) is an inducible transcription factor of the Rel family, and is sequestered in the cytoplasm by the IkappaB family of proteins. NF-kappaB can exist in several dimeric forms, but the p50/p65 heterodimer is the predominant one. Activation of NF-kappaB by a range of stimuli including viral products, and oxidative stress, leads to phosphorylation and proteasome dependent degradation of IkappaB, leading to the release of free NF-kappaB. This free NF-kappaB then binds to its target sites (KB sites in the DNA) to initiate transcription. These kappaB sites are also present in the Long Terminal Repeat (LTR) of HIV-1, and hence NF-kappaB (p50 subunit) binding to LTR-DNA is critical in viral replication. Targeting direct p50-DNA binding, in this regard, is a novel approach to design anti-HIV gene expression inhibitors, which do not have the problem of resistance unlike in other anti-HIV strategies. The present study is a part of our search for leads for the specific inhibition of p50-DNA binding. We have been experimentally studying different types of these inhibitors, and in this work, we attempted to get a common definition of their structural mechanism onto p50-DNA binding. Using three different classes of inhibitors, we modelled their association with the DNA-Binding Region (DBR) of the p50 subunit of NF-kappaB. Docking studies were carried out using a genetic algorithm based program (GOLD). Further, to compare electrostatic complementarity in the association of the inhibitors with the DBR, Molecular Electrostatic Potentials (MEPs) were generated for the DBR and each inhibitor. The results of docking revealed a strong network of hydrogen bonding interactions for every active inhibitor, and the contrary for the less active ones. Further, the MEPs revealed that the DBR of p50 represents a surface of electropositive potential, and the active inhibitors represent a complementary electronegative surface. With the present modelling study we

  10. A molecular modeling study of inhibitors of nuclear factor kappa-B (p50) DNA binding

    NASA Astrophysics Data System (ADS)

    Pande, Vineet; Sharma, Rakesh K.; Inoue, Jun-Ichiro; Otsuka, Masami; Ramos, Maria J.

    2003-12-01

    Nuclear Factor-kappa B (NF-κB) is an inducible transcription factor of the Rel family, and is sequestered in the cytoplasm by the IκB family of proteins. NF-κB can exist in several dimeric forms, but the p50/p65 heterodimer is the predominant one. Activation of NF-κB by a range of stimuli including viral products, and oxidative stress, leads to phosphorylation and proteasome dependent degradation of IκB, leading to the release of free NF-κB. This free NF-κB then binds to its target sites (κB sites in the DNA) to initiate transcription. These κB sites are also present in the Long Terminal Repeat (LTR) of HIV-1, and hence NF-κB (p50 subunit) binding to LTR-DNA is critical in viral replication. Targeting direct p50-DNA binding, in this regard, is a novel approach to design anti-HIV gene expression inhibitors, which do not have the problem of resistance unlike in other anti-HIV strategies. The present study is a part of our search for leads for the specific inhibition of p50-DNA binding. We have been experimentally studying different types of these inhibitors, and in this work, we attempted to get a common definition of their structural mechanism onto p50-DNA binding. Using three different classes of inhibitors, we modelled their association with the DNA-Binding Region (DBR) of the p50 subunit of NF-κB. Docking studies were carried out using a genetic algorithm based program (GOLD). Further, to compare electrostatic complementarity in the association of the inhibitors with the DBR, Molecular Electrostatic Potentials (MEPs) were generated for the DBR and each inhibitor. The results of docking revealed a strong network of hydrogen bonding interactions for every active inhibitor, and the contrary for the less active ones. Further, the MEPs revealed that the DBR of p50 represents a surface of electropositive potential, and the active inhibitors represent a complementary electronegative surface. With the present modelling study we conclude that the principal

  11. Improved coagulation and haemostasis in haemophilia with inhibitors by combinations of superFactor Va and Factor VIIa.

    PubMed

    Bhat, Vikas; von Drygalski, Annette; Gale, Andrew J; Griffin, John H; Mosnier, Laurent O

    2016-03-01

    Bypassing inhibitors in haemophilia patients is limited to activated (a) Factor(F)VII products. We introduced "FVa activity augmentation" as another bypassing strategy and studied effects of an engineered FVa variant designated superFVa. Procoagulant and clot stabilising properties of superFVa and recombinant human (rh)FVIIa, either alone or in combination, were studied in thrombin generation and clot lysis assays in normal human plasma (NHP) with or without anti-FVIII inhibitors, in haemophilia plasma, and in FVIII-deficient mice or in wild-type mice with anti-FVIII inhibitors. SuperFVa was as effective as rhFVIIa to improve thrombin generation or clot lysis. Furthermore, procoagulant effects were significantly enhanced when these compounds were combined. RhFVIIa at 40 nM (a therapeutic concentration) improved thrombin generation mildly, but markedly improved thrombin generation when combined with a low concentration (e. g. 3 nM) of superFVa. In clot lysis studies, the concentration of rhFVIIa to normalise clot lysis times could be reduced by 100-fold (e. g. from 40 nM to 0.4 nM) when combined with a low concentration (0.37 nM) of superFVa. In haemostasis studies of FVIII-deficient mice, blood loss was dose-dependently reduced by either superFVa or rhFVIIa. SuperFVa (200 U/kg) corrected mean blood loss indistinguishably from rhFVIII. Blood loss correction by rhFVIIa was greatly improved when combined with superFVa. Similar blood loss correction results were observed for therapies in wild-type mice after infusion with anti-FVIII inhibitors. Thus, superFVa may be an effective procoagulant agent in the setting of haemophilia with inhibitors and it merits further evaluation for new bypassing strategies.

  12. Improved coagulation and hemostasis in hemophilia with inhibitors by combinations of superFactor Va and Factor VIIa

    PubMed Central

    Bhat, Vikas; von Drygalski, Annette; Gale, Andrew J.; Griffin, John H.; Mosnier, Laurent O.

    2015-01-01

    Bypassing inhibitors in hemophilia patients is limited to activated (a) Factor(F)VII products. We introduced “FVa activity augmentation” as another bypassing strategy and studied effects of an engineered FVa variant designated superFVa. Procoagulant and clot stabilizing properties of superFVa and recombinant human (rh)FVIa, either alone or in combination, were studied in thrombin generation and clot lysis assays in normal human plasma (NHP) with or without anti-FVIII inhibitors, in hemophilia plasma, and in FVIII-deficient mice or in wild-type mice with anti-FVIII inhibitors. superFVa was as effective as rhFVIIa to improve thrombin generation or clot lysis. Furthermore, procoagulant effects were significantly enhanced when these compounds were combined. RhFVIIa at 40 nM (a therapeutic concentration) improved thrombin generation mildly, but markedly improved thrombin generation when combined with a low concentration (e.g., 3 nM) of superFVa. In clot lysis studies, the concentration of rhFVIIa to normalize clot lysis times could be reduced by 100-fold (e.g., from 40 nM to 0.4 nM) when combined with a low concentration (0.37 nM) of superFVa. In hemostasis studies of FVIII-deficient mice, blood loss was dose-dependently reduced by either superFVa or rhFVIIa. SuperFVa (200 U/kg) corrected mean blood loss indistinguishably from rhFVIII. Blood loss correction by rhFVIIa was greatly improved when combined with superFVa. Similar blood loss correction results were observed for therapies in wild-type mice after infusion with anti-FVIII inhibitors. Thus, superFVa may be an effective procoagulant agent in the setting of hemophilia with inhibitors and it merits further evaluation for new bypassing strategies. PMID:26466980

  13. Small-molecule inhibitors of lethal factor protease activity protect against anthrax infection.

    PubMed

    Moayeri, Mahtab; Crown, Devorah; Jiao, Guan-Sheng; Kim, Seongjin; Johnson, Alan; Leysath, Clinton; Leppla, Stephen H

    2013-09-01

    Bacillus anthracis, the causative agent of anthrax, manifests its pathogenesis through the action of two secreted toxins. The bipartite lethal and edema toxins, a combination of lethal factor or edema factor with the protein protective antigen, are important virulence factors for this bacterium. We previously developed small-molecule inhibitors of lethal factor proteolytic activity (LFIs) and demonstrated their in vivo efficacy in a rat lethal toxin challenge model. In this work, we show that these LFIs protect against lethality caused by anthrax infection in mice when combined with subprotective doses of either antibiotics or neutralizing monoclonal antibodies that target edema factor. Significantly, these inhibitors provided protection against lethal infection when administered as a monotherapy. As little as two doses (10 mg/kg) administered at 2 h and 8 h after spore infection was sufficient to provide a significant survival benefit in infected mice. Administration of LFIs early in the infection was found to inhibit dissemination of vegetative bacteria to the organs in the first 32 h following infection. In addition, neutralizing antibodies against edema factor also inhibited bacterial dissemination with similar efficacy. Together, our findings confirm the important roles that both anthrax toxins play in establishing anthrax infection and demonstrate the potential for small-molecule therapeutics targeting these proteins.

  14. Management of cardiac toxicity in patients receiving vascular endothelial growth factor signaling pathway inhibitors.

    PubMed

    Steingart, Richard M; Bakris, George L; Chen, Helen X; Chen, Ming-Hui; Force, Thomas; Ivy, S Percy; Leier, Carl V; Liu, Glenn; Lenihan, Daniel; Lindenfeld, JoAnn; Maitland, Michael L; Remick, Scot C; Tang, W H Wilson

    2012-02-01

    Interfering with angiogenesis is an effective, widely used approach to cancer therapy, but antiangiogenic therapies have been associated with important systemic cardiovascular toxicities such as hypertension, left ventricular dysfunction, heart failure, and myocardial ischemia and infarction. As the use of vascular endothelial growth factor signaling pathway (VSP) inhibitors broadens to include older patients and those with existing cardiovascular disease, the adverse effects are likely to be more frequent, and cardiologists will increasingly be enlisted to help oncologists manage patients who develop adverse cardiovascular effects. The Cardiovascular Toxicities Panel of the National Cancer Institute reviewed the published literature and abstracts from major meetings, shared experience gained during clinical development of VSP inhibitors, and contributed extensive clinical experience in evaluating and treating patients with cancer with cardiovascular disease. This report was edited and approved by the National Cancer Institute Investigational Drug Steering Committee. It presents the panel's expert opinion on the current clinical use and future investigation for safer, more expansive use of these drugs. The panel recommends that physicians (1) conduct and document a formal risk assessment for existing cardiovascular disease and potential cardiovascular complications before VSP inhibitor treatment recognizing that preexisting hypertension and cardiovascular disease are common in patients with cancer, (2) actively monitor for blood pressure elevations and cardiac toxicity with more frequent assessments during the first treatment cycle, and (3) aggressively manage blood pressure elevations and early symptoms and signs of cardiac toxicity to prevent clinically limiting complications of VSP inhibitor therapy. Copyright © 2012 Mosby, Inc. All rights reserved.

  15. Saururus cernuus Lignans - Potent Small Molecule Inhibitors of Hypoxia-Inducible Factor-1

    PubMed Central

    Hossain, Chowdhury Faiz; Kim, Yong-Pil; Baerson, Scott R.; Zhang, Lei; Bruick, Richard K.; Mohammed, Kaleem A.; Agarwal, Ameeta K.; Nagle, Dale G.; Zhou, Yu-Dong

    2010-01-01

    Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B1, manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1α protein accumulation without affecting HIF-1α mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors. PMID:15967416

  16. Saururus cernuus lignans--potent small molecule inhibitors of hypoxia-inducible factor-1.

    PubMed

    Hossain, Chowdhury Faiz; Kim, Yong-Pil; Baerson, Scott R; Zhang, Lei; Bruick, Richard K; Mohammed, Kaleem A; Agarwal, Ameeta K; Nagle, Dale G; Zhou, Yu-Dong

    2005-08-05

    Hypoxia-inducible factor-1 (HIF-1) represents an important tumor-selective therapeutic target for solid tumors. In search of novel small molecule HIF-1 inhibitors, 5400 natural product-rich extracts from plants, marine organisms, and microbes were examined for HIF-1 inhibitory activities using a cell-based reporter assay. Bioassay-guided fractionation and isolation, followed by structure elucidation, yielded three potent natural product-derived HIF-1 inhibitors and two structurally related inactive compounds. In a T47D cell-based reporter assay, manassantin B1, manassantin A, and 4-O-methylsaucerneol inhibited hypoxia-induced HIF-1 activation with IC50 values of 3, 3, and 20 nM, respectively. All three compounds are relatively hypoxia-specific inhibitors of HIF-1 activation, in comparison to other stimuli. The hypoxic induction of HIF-1 target genes CDKN1A, VEGF, and GLUT-1 were also inhibited. These compounds inhibit HIF-1 by blocking hypoxia-induced nuclear HIF-1alpha protein accumulation without affecting HIF-1alpha mRNA levels. In addition, preliminary structure-activity studies suggest specific structural requirements for this class of HIF-1 inhibitors.

  17. RGS Spectroscopy of the Cygnus Loop XA Knot

    NASA Technical Reports Server (NTRS)

    Gaetz, Terrance J.; Mushotzky, Richard F. (Technical Monitor)

    2003-01-01

    The observations were performed at the end of April 2002, and the data were received in July 2002. Unfortunately, the observations were badly compromised by high levels of background radiation; one the three observations lost entirely. Two replacement observations were scheduled for November 2002, and were only made available in January of 2003. Consequently, we have had little time to grapple with the unusual data analysis challenges. The search for a postdoctoral fellow has been successfully concluded, and Manami Sasaki began working for us in January 2003. She will be supported in part by these funds, and will be working to help understand these data. Examination of the RGS 'Orders' images indicate the presence of broad emission lines (as expected for the diffuse XA knot). However, examination of the 'Spatial' dispersion/cross-dispersion images indicate that the emission is also broad in the cross-dispersion direction. (As a crosscheck, some of the 'Lockman Hole' datasets were also examined as representative 'sky background' datasets; in these, both types of images are relatively flat (outside the calibration source regions). The quicklook plots of the spectra show the expected O VII and O VIII lines, in addition to a complex around 35 Angstroms; the approx. 35 Angstrom line is likely the C V He-beta line at 34.97 Angstrom, but identifying the additional line(s) will require a more careful reduction of the data. Consequently, there is valuable information to be extracted from these data, but it is complicated by diffuse nature of the emission. Because the angular scale is large, we will have to make use of sky background datasets in order to do the background fitting. A color composite image of OM data in the three UV bands was presented at the 'How does the Galaxy Work?' meeting, and compared to optical and X-ray imaging data. Quantitative analysis will require obtaining the effective bandpasses of the UV filters so that the predominant line and continuum

  18. Parents' information needs and influential factors when making decisions about TNF-α inhibitors.

    PubMed

    Lipstein, Ellen A; Lovell, Daniel J; Denson, Lee A; Kim, Sandra C; Spencer, Charles; Britto, Maria T

    2016-09-15

    Parents struggle when making treatment decisions for children with arthritis or other chronic conditions. Understanding their decision-making process is an essential step towards improving the decision-making experience. The objective of this study was to describe parents' information needs and the influences on their decision making about treatment with TNF-α inhibitors. Survey domains were developed based on qualitative data and cognitive interviewing. We mailed the survey to parents of children with juvenile idiopathic arthritis or inflammatory bowel disease who had initiated treatment with TNF-α inhibitors in the prior 2 years. Data were analyzed using descriptive and non-parametric statistics. Survey response rate was 54.9 %. Each item had <2 % missing responses. Parents used an array of information sources when deciding about treatment with TNF-α inhibitors. Resources other than their child's specialist were most often used to increase confidence in parents' decisions or because they wanted to know more about other people's experiences being treated with TNF-α inhibitors, rather than due to a lack of understanding. All but two (cost and route of administration) of the influential decision factors were very or extremely important to the majority of participants with factors related to long-term side effects, treatment efficacy, and disease impact being most important. This study describes parents' information needs and influential factors in treatment decision making. Results suggest that future work should be aimed at helping families weigh risks and benefits, such as through decision support interventions, as well as developing opportunities to include people beyond the family and physician in the decision-making process.

  19. Crosstalks between Raf-kinase inhibitor protein and cancer stem cell transcription factors (Oct4, KLF4, Sox2, Nanog).

    PubMed

    Lee, SoHyun; Wottrich, Stephanie; Bonavida, Benjamin

    2017-04-01

    Raf-kinase inhibitor protein has been reported to inhibit both the Raf/mitogen extracellular signal-regulated kinase/extracellular signal-regulated kinase and nuclear factor kappa-light-chain of activated B cells pathways. It has also been reported in cancers that Raf-kinase inhibitor protein behaves as a metastatic suppressor as well as a chemo-immunosensitizing factor to drug/immune-mediated apoptosis. The majority of cancers exhibit low or no levels of Raf-kinase inhibitor protein. Hence, the activities of Raf-kinase inhibitor protein contrast, in part, to those mediated by several cancer stem cell transcription factors for their roles in resistance and metastasis. In this review, the existence of crosstalks in the signaling pathways between Raf-kinase inhibitor protein and several cancer stem cell transcription factors (Oct4, KLF4, Sox2 and Nanog) was assembled. Oct4 is induced by Lin28, and Raf-kinase inhibitor protein inhibits the microRNA binding protein Lin28. The expression of Raf-kinase inhibitor protein inversely correlates with the expression of Oct4. KLF4 does not interact directly with Raf-kinase inhibitor protein, but rather interacts indirectly via Raf-kinase inhibitor protein's regulation of the Oct4/Sox2/KLF4 complex through the mitogen-activated protein kinase pathway. The mechanism by which Raf-kinase inhibitor protein inhibits Sox2 is via the inhibition of the mitogen-activated protein kinase pathway by Raf-kinase inhibitor protein. Thus, Raf-kinase inhibitor protein's relationship with Sox2 is via its regulation of Oct4. Inhibition of extracellular signal-regulated kinase by Raf-kinase inhibitor protein results in the upregulation of Nanog. The inhibition of Oct4 by Raf-kinase inhibitor protein results in the failure of the heterodimer formation of Oct4 and Sox2 that is necessary to bind to the Nanog promoter for the transcription of Nanog. The findings revealed that there exists a direct correlation between the expression of Raf

  20. Risk Factors for Resistance to β-Lactam/β-Lactamase Inhibitors and Ertapenem in Bacteroides Bacteremia.

    PubMed

    Smith, Janessa M; Avdic, Edina; Tamma, Pranita D; Zhang, Long; Carroll, Karen C; Cosgrove, Sara E

    2015-08-01

    The objective of this study was to determine risk factors for the development of resistance to β-lactams/β-lactamase inhibitors (βL/βLIs) and ertapenem among Bacteroides species bacteremia. We conducted a retrospective case-control study of 101 adult patients with Bacteroides species bacteremia at a 1,051-bed tertiary care medical center. The duration of exposure to βL/βLIs (odds ratio [OR], 1.25; 95% confidence interval [CI], 1.08 to 2.31) was the only independent risk factor for resistance. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  1. Proteinuria, a modifiable risk factor: angiotensin converting enzyme inhibitors (ACEIs) and angiotensin II receptor blockers (ARBs).

    PubMed

    Dykeman-Sharpe, Jennifer

    2003-01-01

    Microalbuminuria and proteinuria have been determined to be modifiable risk factors for the progression of chronic kidney disease as well as risk factors for cardiovascular events. Angiotensin converting enzyme inhibitors and angiotensin II receptor blockers have been demonstrated to decrease proteinuria at all stages and slow the progression of renal disease. Proteinuria can be used as a marker of successful treatment in patients with chronic kidney disease in combination with other established targets. This article discusses the various diagnostic tests used for the detection of microalbuminuria and proteinuria and appropriate pharmaceutical treatment.

  2. Tissue Factor Pathway Inhibitor: Multiple Anticoagulant Activities for a Single Protein.

    PubMed

    Mast, Alan E

    2016-01-01

    Tissue factor (TF) pathway inhibitor (TFPI) is an anticoagulant protein that inhibits early phases of the procoagulant response. Alternatively spliced isoforms of TFPI are differentially expressed by endothelial cells and human platelets and plasma. The TFPIβ isoform localizes to the endothelium surface where it is a potent inhibitor of TF-factor VIIa complexes that initiate blood coagulation. The TFPIα isoform is present in platelets. TFPIα contains a stretch of 9 amino acids nearly identical to those found in the B-domain of factor V that are well conserved in mammals. These amino acids provide exosite binding to activated factor V, which allows for TFPIα to inhibit prothrombinase during the initiation phase of blood coagulation. Endogenous inhibition at this point in the coagulation cascade was only recently recognized and has provided a biochemical rationale to explain the pathophysiological mechanisms underlying several clinical disorders. These include the east Texas bleeding disorder that is caused by production of an altered form of factor V with high affinity for TFPI and a paradoxical procoagulant effect of heparins. In addition, these findings have led to ideas for pharmacological targeting of TFPI that may reduce bleeding in hemophilia patients.

  3. Tissue Factor Pathway Inhibitor: Multiple Anticoagulant Activities for a Single Protein

    PubMed Central

    Mast, Alan E.

    2015-01-01

    Tissue Factor Pathway Inhibitor (TFPI) is an anticoagulant protein that inhibits early phases of the procoagulant response. Alternatively spliced isoforms of TFPI are differentially expressed by endothelial cells and human platelets and plasma. The TFPIβ isoform localizes to the endothelium surface where it is a potent inhibitor of tissue factor-factor VIIa complexes that initiate blood coagulation. The TFPIα isoform is present in platelets. TFPIα contains a stretch of 9 amino acids nearly identical to those found in the B-domain of factor V (fV) that are well conserved in mammals. These amino acids provide exosite binding to activated fV, which allows for TFPIα to inhibit prothrombinase during the initiation phase of blood coagulation. Endogenous inhibition at this point in the coagulation cascade was only recently recognized and has provided a biochemical rationale to explain the pathophysiological mechanisms underlying several clinical disorders. These include the east Texas bleeding disorder that is caused by production of an altered form of factor V with high affinity for TFPI, and a paradoxical procoagulant effect of heparins. In addition, these findings have led to ideas for pharmacological targeting of TFPI that may reduce bleeding in hemophilia patients. PMID:26603155

  4. TaXA21-A1 on chromosome 5AL is associated with resistance to multiple pests in wheat.

    PubMed

    Liu, Meiyan; Lei, Lei; Powers, Carol; Liu, Zhiyong; Campbell, Kimberly G; Chen, Xianming; Bowden, Robert L; Carver, Brett F; Yan, Liuling

    2016-02-01

    The wheat ortholog of the rice gene OsXA21 against bacterial leaf blight showed resistance to multiple pests in bread wheat but different interacting proteins. A quantitative trait locus QYr.osu-5A on the long arm of chromosome 5A in bread wheat (Triticum aestivum L., 2n = 6x = 42; AABBDD) was previously reported to confer consistent resistance in adult plants to predominant stripe rust races, but the gene causing the quantitative trait locus (QTL) is not known. Single-nucleotide polymorphism (SNP) markers were used to saturate the QTL region. Comparative and syntenic regions between wheat and rice (Oryza sativa) were applied to identify candidate genes for QYr.osu-5A. TaXA21-A1, which is referred to as a wheat ortholog of OsXA21-like gene on chromosome 9 in rice, was mapped under the peak of the QYr.osu-5A. TaXA21-A1 not only explained the phenotypic variation in reaction to different stripe rust races but also showed significant effects on resistance to powdery mildew and Hessian fly biotype BP. The natural allelic variation resulted in the alternations of four amino acids in deduced TaXA21-A1 proteins. The interacting proteins of TaXA21-A1 were different from those identified by OsXA21 on rice chromosome 11 against bacterial leaf blight. TaXA21-A1 confers unique resistance against multiple pests in wheat but might not have common protein interactors or thus overlapping functions with OsXA21 in rice. XA21 function has diverged during evolution of cereal crops. The molecular marker developed for TaXA21-A1 would accelerate its application of the candidate gene at the QYr.osu-5A locus in wheat breeding programs.

  5. Highly predictive support vector machine (SVM) models for anthrax toxin lethal factor (LF) inhibitors.

    PubMed

    Zhang, Xia; Amin, Elizabeth Ambrose

    2016-01-01

    Anthrax is a highly lethal, acute infectious disease caused by the rod-shaped, Gram-positive bacterium Bacillus anthracis. The anthrax toxin lethal factor (LF), a zinc metalloprotease secreted by the bacilli, plays a key role in anthrax pathogenesis and is chiefly responsible for anthrax-related toxemia and host death, partly via inactivation of mitogen-activated protein kinase kinase (MAPKK) enzymes and consequent disruption of key cellular signaling pathways. Antibiotics such as fluoroquinolones are capable of clearing the bacilli but have no effect on LF-mediated toxemia; LF itself therefore remains the preferred target for toxin inactivation. However, currently no LF inhibitor is available on the market as a therapeutic, partly due to the insufficiency of existing LF inhibitor scaffolds in terms of efficacy, selectivity, and toxicity. In the current work, we present novel support vector machine (SVM) models with high prediction accuracy that are designed to rapidly identify potential novel, structurally diverse LF inhibitor chemical matter from compound libraries. These SVM models were trained and validated using 508 compounds with published LF biological activity data and 847 inactive compounds deposited in the Pub Chem BioAssay database. One model, M1, demonstrated particularly favorable selectivity toward highly active compounds by correctly predicting 39 (95.12%) out of 41 nanomolar-level LF inhibitors, 46 (93.88%) out of 49 inactives, and 844 (99.65%) out of 847 Pub Chem inactives in external, unbiased test sets. These models are expected to facilitate the prediction of LF inhibitory activity for existing molecules, as well as identification of novel potential LF inhibitors from large datasets. Copyright © 2015 Elsevier Inc. All rights reserved.

  6. Recent Development of the Second and Third Generation Irreversible Epidermal Growth Factor Receptor Inhibitors.

    PubMed

    Han, Weiwei; Du, Yongli

    2017-07-01

    Recent reports suggested that essential directions for new lung cancer, breast carcinoma therapies, as well as the roomier realm of targeted cancer therapies were provided through targeting the epidermal growth factor receptor (EGFR). Patients who carrying non-small cell lung carcinoma (NSCLC) with activating mutations in EGFR initially respond well to the EGFR inhibitors erlotinib and gefitinib, which were located the active site of the EGFR kinase and designed to act as competitive inhibitors of combining with the ATP. However, patients who were treated with the erlotinib and gefitinib will relapse because of the emergence of drug-resistant mutations, with T790M mutations accounting for approximately 60% of all resistance. In order to overcome drug resistance, Pharmaceutical chemistry experts recently devoted great endeavors to the development of second-generation irreversible selective inhibitors which covalently modify Cys797 or Cys773 at the ATP binding cleft. Nevertheless, these inhibitors have not reached ideal effect of experts in patients with T790M positive mutation and apparently because of the dose-limiting toxicities associated with inhibition of wild type EGFR. A novel class of 'third generation' EGFR TKIs have been developed that is sensitising and T790M mutant-specific whilst sparing WT EGFR, representing a significant breakthrough in the treatment in NSCLC patients with acquired resistance harboring these genotypes. Herein, we provides an overview of the second and third generation inhibitors currently approved, in clinical trial and also encompasses novel structures of discovery. This review mainly focuses on drug resistance, their mechanisms of action, development of structure-activity relationships and binding modes. © 2017 Wiley-VHCA AG, Zurich, Switzerland.

  7. An alternative inhibitor overcomes resistance caused by a mutation of the epidermal growth factor receptor.

    PubMed

    Kobayashi, Susumu; Ji, Hongbin; Yuza, Yuki; Meyerson, Matthew; Wong, Kwok-Kin; Tenen, Daniel G; Halmos, Balázs

    2005-08-15

    Mutations of the epidermal growth factor receptor (EGFR) gene have been identified in non-small cell lung cancer specimens from patients responding to anilinoquinazoline EGFR inhibitors. However, clinical resistance to EGFR inhibitor therapy is commonly observed. Previously, we showed that such resistance can be caused by a second mutation of the EGFR gene, leading to a T790M amino acid change in the EGFR tyrosine kinase domain and also found that CL-387,785, a specific and irreversible anilinoquinazoline EGFR inhibitor, was able to overcome this resistance on the biochemical level. Here, we present the successful establishment of a stable Ba/F3 cell line model system for the study of oncogenic EGFR signaling and the functional consequences of the EGFR T790M resistance mutation. We show the ability of gefitinib to induce growth arrest and apoptosis in cells transfected with wild-type or L858R EGFR, whereas the T790M mutation leads to high-level functional resistance against gefitinib and erlotinib. In addition, CL-387,785 is able to overcome resistance caused by the T790M mutation on a functional level, correlating with effective inhibition of downstream signaling pathways. Similar data was also obtained with the use of the gefitinib-resistant H1975 lung cancer cell line. The systems established by us should prove useful for the large-scale screening of alternative EGFR inhibitor compounds against the T790M or other EGFR mutations. These data also support the notion that clinical investigations of compounds similar to CL-387,785 may be useful as a treatment strategy for patients with resistance to EGFR inhibitor therapy caused by the T790M mutation.

  8. A Novel Malate Dehydrogenase 2 Inhibitor Suppresses Hypoxia-Inducible Factor-1 by Regulating Mitochondrial Respiration

    PubMed Central

    Jang, Kusik; Kim, Inhyub; Kim, Bo-Kyung; Lee, Kyeong; Won, Misun

    2016-01-01

    We previously reported that hypoxia-inducible factor (HIF)-1 inhibitor LW6, an aryloxyacetylamino benzoic acid derivative, inhibits malate dehydrogenase 2 (MDH2) activity during the mitochondrial tricarboxylic acid (TCA) cycle. In this study, we present a novel MDH2 inhibitor compound 7 containing benzohydrazide moiety, which was identified through structure-based virtual screening of chemical library. Similar to LW6, compound 7 inhibited MDH2 activity in a competitive fashion, thereby reducing NADH level. Consequently, compound 7 reduced oxygen consumption and ATP production during the mitochondrial respiration cycle, resulting in increased intracellular oxygen concentration. Therefore, compound 7 suppressed the accumulation of HIF-1α and expression of its target genes, vascular endothelial growth factor (VEGF) and glucose transporter 1 (GLUT1). Moreover, reduction in ATP content activated AMPK, thereby inactivating ACC and mTOR the downstream pathways. As expected, compound 7 exhibited significant growth inhibition of human colorectal cancer HCT116 cells. Compound 7 demonstrated substantial anti-tumor efficacy in an in vivo xenograft assay using HCT116 mouse model. Taken together, a novel MDH2 inhibitor, compound 7, suppressed HIF-1α accumulation via reduction of oxygen consumption and ATP production, integrating metabolism into anti-cancer efficacy in cancer cells. PMID:27611801

  9. Identification of inhibitors of a bacterial sigma factor using a new high-throughput screening assay.

    PubMed

    El-Mowafi, S A; Sineva, E; Alumasa, J N; Nicoloff, H; Tomsho, J W; Ades, S E; Keiler, K C

    2015-01-01

    Gram-negative bacteria are formidable pathogens because their cell envelope presents an adaptable barrier to environmental and host-mediated challenges. The stress response pathway controlled by the alternative sigma factor σ(E) is critical for maintenance of the cell envelope. Because σ(E) is required for the virulence or viability of several Gram-negative pathogens, it might be a useful target for antibiotic development. To determine if small molecules can inhibit the σ(E) pathway, and to permit high-throughput screening for antibiotic lead compounds, a σ(E) activity assay that is compatible with high-throughput screening was developed and validated. The screen employs a biological assay with positive readout. An Escherichia coli strain was engineered to express yellow fluorescent protein (YFP) under negative regulation by the σ(E) pathway, such that inhibitors of the pathway increase the production of YFP. To validate the screen, the reporter strain was used to identify σ(E) pathway inhibitors from a library of cyclic peptides. Biochemical characterization of one of the inhibitory cyclic peptides showed that it binds σ(E), inhibits RNA polymerase holoenzyme formation, and inhibits σ(E)-dependent transcription in vitro. These results demonstrate that alternative sigma factors can be inhibited by small molecules and enable high-throughput screening for inhibitors of the σ(E) pathway.

  10. Rational design of potent and selective inhibitors of an epoxide hydrolase virulence factor from Pseudomonas aeruginosa

    PubMed Central

    Kitamura, Seiya; Hvorecny, Kelli L.; Niu, Jun; Hammock, Bruce D.; Madden, Dean R.; Morisseau, Christophe

    2016-01-01

    The virulence factor cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is secreted by Pseudomonas aeruginosa and is the founding member of a distinct class of epoxide hydrolases (EHs) that triggers the catalysis-dependent degradation of the CFTR. We describe here the development of a series of potent and selective Cif inhibitors by structure-based drug design. Initial screening revealed 1a (KB2115), a thyroid hormone analog, as a lead compound with low micromolar potency. Structural requirements for potency were systematically probed, and interactions between Cif and 1a were characterized by X-ray crystallography. Based on these data, new compounds were designed to yield additional hydrogen bonding with residues of the Cif active site. From this effort, three compounds were identified that are 10-fold more potent toward Cif than our first-generation inhibitors and have no detectable thyroid hormone-like activity. These inhibitors will be useful tools to study the pathological role of Cif, and have the potential for clinical application. PMID:27120257

  11. Rational Design of Potent and Selective Inhibitors of an Epoxide Hydrolase Virulence Factor from Pseudomonas aeruginosa.

    PubMed

    Kitamura, Seiya; Hvorecny, Kelli L; Niu, Jun; Hammock, Bruce D; Madden, Dean R; Morisseau, Christophe

    2016-05-26

    The virulence factor cystic fibrosis transmembrane conductance regulator (CFTR) inhibitory factor (Cif) is secreted by Pseudomonas aeruginosa and is the founding member of a distinct class of epoxide hydrolases (EHs) that triggers the catalysis-dependent degradation of the CFTR. We describe here the development of a series of potent and selective Cif inhibitors by structure-based drug design. Initial screening revealed 1a (KB2115), a thyroid hormone analog, as a lead compound with low micromolar potency. Structural requirements for potency were systematically probed, and interactions between Cif and 1a were characterized by X-ray crystallography. On the basis of these data, new compounds were designed to yield additional hydrogen bonding with residues of the Cif active site. From this effort, three compounds were identified that are 10-fold more potent toward Cif than our first-generation inhibitors and have no detectable thyroid hormone-like activity. These inhibitors will be useful tools to study the pathological role of Cif and have the potential for clinical application.

  12. Identification and analysis of the tissue factor pathway inhibitor 2 of Sciaenops ocellatus.

    PubMed

    Zhang, Min; Xiao, Zhi-zhong; Sun, Li

    2011-01-01

    Tissue factor pathway inhibitor 2 (TFPI-2) is a structural homologue of TFPI, a potent inhibitor of tissue factor (TF)-mediated coagulation. Although TFPI-2 has been identified at sequence level in several fish species, no study on piscine TFPI-2 has been documented. In this report, we identified and analyzed a TFPI-2 homologue, SoTFPI2, from red drum Sciaenops ocellatus. The open reading frame of SoTFPI2 is 681 bp, which encodes a 226-residue protein that shares 59.2-82.3% overall sequence identities with known fish TFPI-2. SoTFPI2 possesses three tandem Kunitz domains and is negatively charged at the N-terminus and positively charged at the C-terminus. Expression of SoTFPI2 was detected, in increasing order, in spleen, muscle, gill, brain, liver, kidney, blood, and heart. Bacterial challenge and lipopolysaccharide treatment significantly upregulated SoTFPI2 expression in kidney in time-dependent manners. Recombinant SoTFPI2 purified from Escherichia coli inhibits the proteolytic activity of trypsin and exhibits bactericidal effect on a fish pathogen. Take together, these results indicate that SoTFPI2 is a biologically active serine protease inhibitor with antibacterial property and is likely to play a role in anti-bacterial infection.

  13. Comprehensive inhibitor profiling of the Proteus mirabilis metalloprotease virulence factor ZapA (mirabilysin).

    PubMed

    Carson, Louise; Cathcart, George R; Scott, Christopher J; Hollenberg, Morley D; Walker, Brian; Ceri, Howard; Gilmore, Brendan F

    2011-10-01

    In this study we report for the first time the comprehensive inhibitor profiling of the Proteus mirabilis metalloprotease virulence factor ZapA (mirabilysin) using a 160 compound focused library of N-alpha mercaptoamide dipeptides, in order to map the S(1)(') and S(2)(') binding site preferences of this important enzyme. This study has revealed a preference for the aromatic residues tyrosine and tryptophan in P(1)(') and aliphatic residues in P(2)('). From this library, six compounds were identified which exhibited sub- to low-micromolar K(i) values. The most potent inactivator, SH-CO(2)-Y-V-NH(2) was capable of preventing ZapA-mediated hydrolysis of heat-denatured IgA, indicating that these inhibitors may be capable of protecting host proteins against ZapA during colonisation and infection. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  14. Hypoxia inducible factor 1α expression and effects of its inhibitors in canine lymphoma

    PubMed Central

    KAMBAYASHI, Satoshi; IGASE, Masaya; KOBAYASHI, Kosuke; KIMURA, Ayana; SHIMOKAWA MIYAMA, Takako; BABA, Kenji; NOGUCHI, Shunsuke; MIZUNO, Takuya; OKUDA, Masaru

    2015-01-01

    Hypoxic conditions in various cancers are believed to relate with their malignancy, and hypoxia inducible factor-1α (HIF-1α) has been shown to be a major regulator of the response to low oxygen. In this study, we examined HIF-1α expression in canine lymphoma using cell lines and clinical samples and found that these cells expressed HIF-1α. Moreover, the HIF-1α inhibitors, echinomycin, YC-1 and 2-methoxyestradiol, suppressed the proliferation of canine lymphoma cell lines. In a xenograft model using NOD/scid mice, echinomycin treatment resulted in a dose-dependent regression of the tumor. Our results suggest that HIF-1α contributes to the proliferation and/or survival of canine lymphoma cells. Therefore, HIF-1α inhibitors may be potential agents to treat canine lymphoma. PMID:26050843

  15. Mutation of the rice XA21 predicted nuclear localization sequence does not affect resistance to Xanthomonas oryzae pv. oryzae

    SciTech Connect

    Wei, Tong; Chen, Tsung-Chi; Ho, Yuen Ting; Ronald, Pamela C.

    2016-10-05

    Background: The rice receptor kinase XA21 confers robust resistance to the bacterial pathogenXanthomonas oryzae pv.oryzae(Xoo). We previously reported that XA21 is cleaved in transgenic plants overexpressing XA21 with a GFP tag (Ubi-XA21-GFP) and that the released C-terminal domain is localized to the nucleus. XA21 carries a predicted nuclear localization sequence (NLS) that directs the C-terminal domain to the nucleus in transient assays, whereas alanine substitutions in the NLS disrupt the nuclear localization. Methods: To determine if the predicted NLS is required for XA21-mediated immunityin planta, we generated transgenic plants overexpressing an XA21 variant carrying the NLS with the same alanine substitutions (Ubi-XA21nls-GFP). Results: Ubi- XA21nls-GFP plants displayed slightly longer lesion lengths, higherXoo bacterial populations after inoculation and lower levels of reactive oxygen species production compared with the Ubi- XA21-GFP control plants. However, the Ubi- XA21nls-GFP plants express lower levels of protein than that observed inUbi- XA21-GFP. Discussion: These results demonstrate that the predicted NLS is not required for XA21-mediated immunity.

  16. Tumor necrosis factor-alpha inhibitors suppress CCL2 chemokine in monocytes via epigenetic modification.

    PubMed

    Lin, Yi-Ching; Lin, Yu-Chih; Huang, Ming-Yii; Kuo, Po-Lin; Wu, Cheng-Chin; Lee, Min-Sheng; Hsieh, Chong-Chao; Kuo, Hsuan-Fu; Kuo, Chang-Hung; Tsai, Wen-Chan; Hung, Chih-Hsing

    2017-03-01

    The treatment of rheumatoid arthritis (RA) with tumor necrosis factor-alpha (TNF-α) inhibitors could lead to adverse effects. Therefore, the identification of downstream therapeutic targets is important. Monocyte chemoattractant protein-1 (MCP-1, also called CCL2) is related to RA disease activity, and epigenetic modifications are hypothesized to regulate gene expression in RA pathogenesis. We studied the effects of two TNF-α inhibitors, etanercept and adalimumab, on CCL2 expression and the potentially associated intracellular mechanisms, including epigenetic regulation. Etanercept and adalimumab decreased CCL2 production in THP-1 cells and human primary monocytes, as detected using enzyme-linked immunosorbent assays, and these changes in the CCL2 levels were independent of the TNF-α levels. Etanercept and adalimumab suppressed mitogen-activated protein kinase (MAPK) phospho-p38, phospho-JNK, phospho-ERK and nuclear factor-κB (NF-κB) phospho-p65, as demonstrated using western blot analyses. The investigation of epigenetic modifications using chromatin immunoprecipitation revealed that etanercept and adalimumab down-regulated acetylation of histone (H)3 and H4 in the CCL2 promoter region by decreasing the recruitment of the NF-κB associated acetyltransferases p300, CBP and PCAF. Etanercept and adalimumab also down-regulated trimethylation of H3K4, H3K27, H3K36 and H3K79 in the CCL2 promoter region by decreasing the expression of the related methyltransferases WDR5 and Smyd2. We demonstrated that TNF-α inhibitors exert immunomodulatory effects on CCL2 expression in human monocytes via MAPKs, NF-κB and epigenetic modifications. These findings broaden the mechanistic knowledge related to TNF-α inhibitors and provide novel therapeutic targets for RA.

  17. Antiplatelet agents affecting the interaction of Tissue Factor-Factor VIIa complex with Factor X in a continuous-flow reactor.

    PubMed

    Gir, S; Reavis, R; Turitto, V T; Gollamudi, R

    1996-02-01

    The purpose of the present study was to examine the role of antithrombotic agents in the activation of Factor X in the presence of the Tissue Factor-Factor VIIa (TF-VIIa) complex in a continuous-flow reactor. Tissue Factor immobilized in a phospholipid bilayer on the inner surface of a capillary tube (internal diameter = 0.27 mm) was exposed to a perfusate containing Factors VIIa and X flowing at a flow rate of 12.7 microliters/min, corresponding to a wall shear rate of 100 s-1. Factor Xa (the activated form of Factor X) in the effluent was determined by a chromogenic assay. The effectiveness of two platelet aggregation inhibitors, alpha,alpha'-bis-[3-(N,N-diethylcarbamoyl)piperidino-p-xylene dihydrobromide (A-1) and alpha,alpha'-bis-[3-N-benzyl-N-methylcarbamoyl)piperidino]-p-xylen e dihydrobromide (A-4) in inhibiting Factor X activation is reported here. The results suggest that the Tissue Factor pathway, mediated through TF-VIIa complex, produces significantly lower levels of Factor Xa in the presence of compounds A-1 and A-4. On the basis of these findings, it appears that the anticoagulation action of these compounds reinforces their platelet aggregation-inhibitory properties. These carbamoylpiperidines (nipecotamides) therefore appear to be useful antithrombotic agents.

  18. Tissue factor and tissue factor pathway inhibitor as key regulators of global hemostasis: measurement of their levels in coagulation assays.

    PubMed

    Kasthuri, Raj S; Glover, Sam L; Boles, Jeremiah; Mackman, Nigel

    2010-10-01

    The tissue factor (TF)/factor (F)VIIa complex is the primary initiator of coagulation in vivo. Tissue factor pathway inhibitor (TFPI) is the physiological inhibitor of the TF/FVIIa complex. Deficiencies of either TF or TFPI have not been reported in humans, and a complete absence of either of these two proteins in mice is embryonically lethal. To maintain normal hemostasis, levels of TF and TFPI need to be balanced. Increased levels of TF can overwhelm the inhibitory capacity of TFPI, resulting in thrombosis. Decreased levels of TF are associated with bleeding. Global assays of coagulation are defined as tests capable of evaluating all components of the clotting cascade that are present in plasma. In these tests the thrombogenic surface is either provided by platelets or exogenous phospholipids. Clotting assays currently used in clinical practice are not designed to measure endogenous levels of TF and TFPI. Therefore, there is a need to develop sensitive and specific assays for measuring levels of functional TF and TFPI in whole blood and plasma. These assays could be useful in patient management in many scenarios.

  19. Activated factor XI increases the procoagulant activity of the extrinsic pathway by inactivating tissue factor pathway inhibitor

    PubMed Central

    Tucker, Erik I.; Matafonov, Anton; Cheng, Qiufang; Zientek, Keith D.; Gailani, Dave; Gruber, András; McCarty, Owen J. T.

    2015-01-01

    Activation of coagulation factor XI (FXI) may play a role in hemostasis. The primary substrate of activated FXI (FXIa) is FIX, leading to FX activation (FXa) and thrombin generation. However, recent studies suggest the hemostatic role of FXI may not be restricted to the activation of FIX. We explored whether FXI could interact with and inhibit the activity of tissue factor pathway inhibitor (TFPI). TFPI is an essential reversible inhibitor of activated factor X (FXa) and also inhibits the FVIIa-TF complex. We found that FXIa neutralized both endothelium- and platelet-derived TFPI by cleaving the protein between the Kunitz (K) 1 and K2 domains (Lys86/Thr87) and at the active sites of the K2 (Arg107/Gly108) and K3 (Arg199/Ala200) domains. Addition of FXIa to plasma was able to reverse the ability of TFPI to prolong TF-initiated clotting times in FXI- or FIX-deficient plasma, as well as FXa-initiated clotting times in FX-deficient plasma. Treatment of cultured endothelial cells with FXIa increased the generation of FXa and promoted TF-dependent fibrin formation in recalcified plasma. Together, these results suggest that the hemostatic role of FXIa may be attributed not only to activation of FIX but also to promoting the extrinsic pathway of thrombin generation through inactivation of TFPI. PMID:25587039

  20. Tissue Factor and Tissue Factor Pathway Inhibitor as Key Regulators of Global Hemostasis: Measurement of Their Levels in Coagulation Assays

    PubMed Central

    Kasthuri, Raj S.; Glover, Sam L.; Boles, Jeremiah; Mackman, Nigel

    2011-01-01

    The tissue factor (TF)/factor (F)VIIa complex is the primary initiator of coagulation in vivo. Tissue factor pathway inhibitor (TFPI) is the physiological inhibitor of the TF/FVIIa complex. Deficiencies of either TF or TFPI have not been reported in humans, and a complete absence of either of these two proteins in mice is embryonically lethal. To maintain normal hemostasis, levels of TF and TFPI need to be balanced. Increased levels of TF can overwhelm the inhibitory capacity of TFPI, resulting in thrombosis. Decreased levels of TF are associated with bleeding. Global assays of coagulation are defined as tests capable of evaluating all components of the clotting cascade that are present in plasma. In these tests the thrombogenic surface is either provided by platelets or exogenous phospholipids. Clotting assays currently used in clinical practice are not designed to measure endogenous levels of TF and TFPI. Therefore, there is a need to develop sensitive and specific assays for measuring levels of functional TF and TFPI in whole blood and plasma. These assays could be useful in patient management in many scenarios. PMID:20978997

  1. Hepatocyte growth factor activator is a potential target proteinase for Kazal-type inhibitor in turkey (Meleagris gallopavo) seminal plasma.

    PubMed

    Słowińska, Mariola; Bukowska, Joanna; Hejmej, Anna; Bilińska, Barbara; Kozłowski, Krzysztof; Jankowski, Jan; Ciereszko, Andrzej

    2015-08-01

    A peculiar characteristic of turkey seminal plasma is the increased activity of serine proteinases. It is of interest if the single-domain Kazal-type inhibitor controls the activity of turkey seminal plasma proteinases. Pure preparations of the Kazal-type inhibitor and anti-Kazal-type inhibitor monospecific immunoglobulin Gs were used as ligands in affinity chromatography for proteinase isolation from turkey seminal plasma. Gene expression and the immunohistochemical detection of the single-domain Kazal-type inhibitor in the reproductive tract of turkey toms are described. The hepatocyte growth factor activator (HGFA) was identified in the binding fraction in affinity chromatography. Hepatocyte growth factor activator activity was inhibited by the Kazal-type inhibitor in a dose-dependent manner. This protease was a primary physiological target for the single-domain Kazal-type inhibitor. Numerous proteoforms of HGFA were present in turkey seminal plasma, and phosphorylation was the primary posttranslational modification of HGFA. In addition to HGFA, acrosin was a target proteinase for the single-domain Kazal-type inhibitor. In seminal plasma, acrosin was present only in complexes with the Kazal-type inhibitor and was not present as a free enzyme. The single-domain Kazal-type inhibitor was specific for the reproductive tract. The germ cell-specific expression of Kazal-type inhibitors in the testis indicated an important function in spermatogenesis; secretion by the epithelial cells of the epididymis and the ductus deferens indicated that the Kazal-type inhibitor was an important factor involved in the changes in sperm membranes during maturation and in the maintenance of the microenvironment in which sperm maturation occurred and sperm was stored. The role of HGFA in these processes remains to be established.

  2. Characterization and expression analysis of two cDNAs encoding Xa1 and oxysterol binding proteins in sorghum (Sorghum bicolor)

    USDA-ARS?s Scientific Manuscript database

    Using suppression subtractive hybridization (SSH) and subsequent microarray analysis, expression profiles of sorghum genes responsive to greenbug phloem-feeding were obtained and identified. Among the profiles, two cDNAs designated to MM73 and MM95 were identified to encode Xa1 (Xa1) and oxysterol ...

  3. Rice Xb15, a protein phosphatase 2C, negatively regulates Xa21-mediated resistance and programmed cell death

    USDA-ARS?s Scientific Manuscript database

    The rice pathogen recognition receptor, XA21, confers resistance to specific races of Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight disease. A yeast-two-hybrid screen using the intracellular portion of XA21, including the juxtamembrane (JM) and kinase domain as a bait, i...

  4. Genetic engineering of the Xa10 promoter for broad-spectrum and durable resistance to Xanthomonas oryzae pv. oryzae.

    PubMed

    Zeng, Xuan; Tian, Dongsheng; Gu, Keyu; Zhou, Zhiyun; Yang, Xiaobei; Luo, Yanchang; White, Frank F; Yin, Zhongchao

    2015-09-01

    Many pathovars of plant pathogenic bacteria Xanthomonas species inject transcription activator-like (TAL) effectors into plant host cells to promote disease susceptibility or trigger disease resistance. The rice TAL effector-dependent disease resistance gene Xa10 confers narrow-spectrum race-specific resistance to Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial blight disease in rice. To generate broad-spectrum and durable resistance to Xoo, we developed a modified Xa10 gene, designated as Xa10(E5) . Xa10(E5) has an EBE-amended promoter containing 5 tandemly arranged EBEs each responding specifically to a corresponding virulent or avirulent TAL effector and a stable transgenic rice line containing Xa10(E5) was generated in the cultivar Nipponbare. The Xa10(E5) gene was specifically induced by Xoo strains that harbour the corresponding TAL effectors and conferred TAL effector-dependent resistance to the pathogens at all developmental stages of rice. Further disease evaluation demonstrated that the Xa10(E5) gene in either Nipponbare or 9311 genetic backgrounds provided broad-spectrum disease resistance to 27 of the 28 Xoo strains collected from 11 countries. The development of Xa10(E5) and transgenic rice lines provides new genetic materials for molecular breeding of rice for broad-spectrum and durable disease resistance to bacterial blight.

  5. Resistance of human glioblastoma multiforme cells to growth factor inhibitors is overcome by blockade of inhibitor of apoptosis proteins

    PubMed Central

    Ziegler, David S.; Wright, Renee D.; Kesari, Santosh; Lemieux, Madeleine E.; Tran, Mary A.; Jain, Monish; Zawel, Leigh; Kung, Andrew L.

    2008-01-01

    Multiple receptor tyrosine kinases (RTKs), including PDGFR, have been validated as therapeutic targets in glioblastoma multiforme (GBM), yet inhibitors of RTKs have had limited clinical success. As various antiapoptotic mechanisms render GBM cells resistant to chemo- and radiotherapy, we hypothesized that these antiapoptotic mechanisms also confer resistance to RTK inhibition. We found that in vitro inhibition of PDGFR in human GBM cells initiated the intrinsic pathway of apoptosis, as evidenced by mitochondrial outer membrane permeabilization, but downstream caspase activation was blocked by inhibitor of apoptosis proteins (IAPs). Consistent with this, inhibition of PDGFR combined with small molecule inactivation of IAPs induced apoptosis in human GBM cells in vitro and had synergistic antitumor effects in orthotopic mouse models of GBM and in primary human GBM neurospheres. These results demonstrate that concomitant inhibition of IAPs can overcome resistance to RTK inhibitors in human malignant GBM cells, and suggest that blockade of IAPs has the potential to improve treatment outcomes in patients with GBM. PMID:18677408

  6. Design, Synthesis, and Protein Crystallography of Biaryltriazoles as Potent Tautomerase Inhibitors of Macrophage Migration Inhibitory Factor

    PubMed Central

    Dziedzic, Pawel; Cisneros, José A.; Robertson, Michael J.; Hare, Alissa A.; Danford, Nadia E.; Baxter, Richard H. G.; Jorgensen, William L.

    2015-01-01

    Optimization is reported for biaryltriazoles as inhibitors of the tautomerase activity of human macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with numerous inflammatory diseases and cancer. A combined approach was taken featuring organic synthesis, enzymatic assaying, crystallography, and modeling including free-energy perturbation (FEP) calculations. X-ray crystal structures for 3a and 3b bound to MIF are reported and provided a basis for the modeling efforts. The accommodation of the inhibitors in the binding site is striking with multiple hydrogen bonds and aryl–aryl interactions. Additional modeling encouraged pursuit of 5-phenoxyquinolinyl analogues, which led to the very potent compound 3s. Activity was further enhanced by addition of a fluorine atom adjacent to the phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. It is also shown that physical properties of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13. PMID:25697265

  7. Small-molecule factor D inhibitors targeting the alternative complement pathway.

    PubMed

    Maibaum, Jürgen; Liao, Sha-Mei; Vulpetti, Anna; Ostermann, Nils; Randl, Stefan; Rüdisser, Simon; Lorthiois, Edwige; Erbel, Paul; Kinzel, Bernd; Kolb, Fabrice A; Barbieri, Samuel; Wagner, Julia; Durand, Corinne; Fettis, Kamal; Dussauge, Solene; Hughes, Nicola; Delgado, Omar; Hommel, Ulrich; Gould, Ty; Mac Sweeney, Aengus; Gerhartz, Bernd; Cumin, Frederic; Flohr, Stefanie; Schubart, Anna; Jaffee, Bruce; Harrison, Richard; Risitano, Antonio Maria; Eder, Jörg; Anderson, Karen

    2016-12-01

    Complement is a key component of the innate immune system, recognizing pathogens and promoting their elimination. Complement component 3 (C3) is the central component of the system. Activation of C3 can be initiated by three distinct routes-the classical, the lectin and the alternative pathways-with the alternative pathway also acting as an amplification loop for the other two pathways. The protease factor D (FD) is essential for this amplification process, which, when dysregulated, predisposes individuals to diverse disorders including age-related macular degeneration and paroxysmal nocturnal hemoglobinuria (PNH). Here we describe the identification of potent and selective small-molecule inhibitors of FD. These inhibitors efficiently block alternative pathway (AP) activation and prevent both C3 deposition onto, and lysis of, PNH erythrocytes. Their oral administration inhibited lipopolysaccharide-induced AP activation in FD-humanized mice. These data demonstrate the feasibility of inhibiting the AP with small-molecule antagonists and support the development of FD inhibitors for the treatment of complement-mediated diseases.

  8. Widespread potential for growth-factor-driven resistance to anticancer kinase inhibitors

    PubMed Central

    Wilson, Timothy R.; Fridlyand, Jane; Yan, Yibing; Penuel, Elicia; Burton, Luciana; Chan, Emily; Peng, Jing; Lin, Eva; Wang, Yulei; Sosman, Jeff; Ribas, Antoni; Li, Jiang; Moffat, John; Sutherlin, Daniel P.; Koeppen, Hartmut; Merchant, Mark; Neve, Richard; Settleman, Jeff

    2013-01-01

    Mutationally activated kinases define a clinically validated class of targets for cancer drug therapy1. However, the efficacy of kinase inhibitors in patients whose tumours harbour such alleles is invariably limited by innate or acquired drug resistance2,3. The identification of resistance mechanisms has revealed a recurrent theme—the engagement of survival signals redundant to those transduced by the targeted kinase4. Cancer cells typically express multiple receptor tyrosine kinases (RTKs) that mediate signals that converge on common critical downstream cell-survival effectors—most notably, phosphatidylinositol-3-OH kinase (PI(3)K) and mitogen-activated protein kinase (MAPK)5. Consequently, an increase in RTK-ligand levels, through autocrine tumour-cell production, paracrine contribution from tumour stroma6 or systemic production, could confer resistance to inhibitors of an oncogenic kinase with a similar signalling output. Here, using a panel of kinase-‘addicted’ human cancer cell lines, we found that most cells can be rescued from drug sensitivity by simply exposing them to one or more RTK ligands. Among the findings with clinical implications was the observation that hepatocyte growth factor (HGF) confers resistance to the BRAF inhibitor PLX4032 (vemurafenib) in BRAF-mutant melanoma cells. These observations highlight the extensive redundancy of RTK-transduced signalling in cancer cells and the potentially broad role of widely expressed RTK ligands in innate and acquired resistance to drugs targeting oncogenic kinases. PMID:22763448

  9. Semiology of skin toxicity associated with epidermal growth factor receptor (EGFR) inhibitors.

    PubMed

    Peuvrel, L; Bachmeyer, C; Reguiai, Z; Bachet, J B; André, T; Bensadoun, R J; Bouché, O; Ychou, M; Dréno, B

    2012-05-01

    Advances in the understanding of the mechanisms involved in oncogenesis have led to the development of so-called targeted therapies such as epidermal growth factor receptor (EGFR) inhibitors, which take on an increasingly important role in the management of cancer. These treatments have the advantage not to trigger the adverse effects traditionally encountered with chemotherapy, such as nausea, vomiting or haematological toxicity. However, they do cause new forms of toxicity: the most common one is skin toxicity. It is important to be aware of it because it can be debilitating, adversely impacting patients' quality of life and altering treatment compliance, although it appears to be correlated with treatment response in certain series. Non-specialists can have difficulty in recognising this unusual skin toxicity. The dermatologic side effects most frequently triggered by EGFR inhibitors are discussed in this article. They are divided into three categories depending on their target: inflammation of the pilo-sebaceous follicle, represented by EGFR inhibitor-associated folliculitis, which occurs at an early stage and is frequent; alteration in the skin barrier, primarily responsible for xerosis, fissures and pruritus, which are frequent and delayed; and lesions of the skin appendages (paronychia, pyogenic granuloma, hair changes), which are delayed and less frequent. It is essential for all practitioners concerned to know about these dermatologic side effects in order to ensure better global management of patients, particularly in terms of quality of life.

  10. Design, Synthesis, and Protein Crystallography of Biaryltriazoles as Potent Tautomerase Inhibitors of Macrophage Migration Inhibitory Factor

    SciTech Connect

    Dziedzic, Pawel; Cisneros, José A.; Robertson, Michael J.; Hare, Alissa A.; Danford, Nadia E.; Baxter, Richard H. G.; Jorgensen, William L.

    2015-02-20

    Optimization is reported for biaryltriazoles as inhibitors of the tautomerase activity of human macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with numerous inflammatory diseases and cancer. A combined approach was taken featuring organic synthesis, enzymatic assaying, crystallography, and modeling including free-energy perturbation (FEP) calculations. X-ray crystal structures for 3a and 3b bound to MIF are reported and provided a basis for the modeling efforts. The accommodation of the inhibitors in the binding site is striking with multiple hydrogen bonds and aryl–aryl interactions. Additional modeling encouraged pursuit of 5-phenoxyquinolinyl analogues, which led to the very potent compound 3s. Activity was further enhanced by addition of a fluorine atom adjacent to the phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. We also show that physical properties of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13.

  11. Design, Synthesis, and Protein Crystallography of Biaryltriazoles as Potent Tautomerase Inhibitors of Macrophage Migration Inhibitory Factor

    DOE PAGES

    Dziedzic, Pawel; Cisneros, José A.; Robertson, Michael J.; ...

    2015-02-20

    Optimization is reported for biaryltriazoles as inhibitors of the tautomerase activity of human macrophage migration inhibitory factor (MIF), a proinflammatory cytokine associated with numerous inflammatory diseases and cancer. A combined approach was taken featuring organic synthesis, enzymatic assaying, crystallography, and modeling including free-energy perturbation (FEP) calculations. X-ray crystal structures for 3a and 3b bound to MIF are reported and provided a basis for the modeling efforts. The accommodation of the inhibitors in the binding site is striking with multiple hydrogen bonds and aryl–aryl interactions. Additional modeling encouraged pursuit of 5-phenoxyquinolinyl analogues, which led to the very potent compound 3s. Activitymore » was further enhanced by addition of a fluorine atom adjacent to the phenolic hydroxyl group as in 3w, 3z, 3aa, and 3bb to strengthen a key hydrogen bond. We also show that physical properties of the compounds can be modulated by variation of solvent-exposed substituents. Several of the compounds are likely the most potent known MIF tautomerase inhibitors; the most active ones are more than 1000-fold more active than the well-studied (R)-ISO-1 and more than 200-fold more active than the chromen-4-one Orita-13.« less

  12. Synthetic peptides and fluorogenic substrates related to the reactive site sequence of Kunitz-type inhibitors isolated from Bauhinia: interaction with human plasma kallikrein.

    PubMed

    Oliva, M L; Santomauro-Vaz, E M; Andrade, S A; Juliano, M A; Pott, V J; Sampaio, M U; Sampaio, C A

    2001-01-01

    We have previously described Kunitz-type serine proteinase inhibitors purified from Bauhinia seeds. Human plasma kallikrein shows different susceptibility to those inhibitors. In this communication, we describe the interaction of human plasma kallikrein with fluorogenic and non-fluorogenic peptides based on the Bauhinia inhibitors' reactive site. The hydrolysis of the substrate based on the B. variegata inhibitor reactive site sequence, Abz-VVISALPRSVFIQ-EDDnp (Km 1.42 microM, kcat 0.06 s(-1), and kcat/Km 4.23 x 10(4) M(-1) s(-1)), is more favorable than that of Abz-VMIAALPRTMFIQ-EDDnp, related to the B. ungulata sequence (Km 0.43 microM, kcat 0.00017 s(-1), and kcat/Km 3.9 x 10(2) M(-1) s(-1)). Human plasma kallikrein does not hydrolyze the substrates Abz-RPGLPVRFESPL-EDDnp and Abz-FESPLRINIIKE-EDDnp based on the B. bauhinioides inhibitor reactive site sequence, the most effective inhibitor of the enzyme. These peptides are competitive inhibitors with Ki values in the nM range. The synthetic peptide containing 19 amino acids based on the B. bauhinioides inhibitor reactive site (RPGLPVRFESPL) is poorly cleaved by kallikrein. The given substrates are highly specific for trypsin and chymotrypsin hydrolysis. Other serine proteinases such as factor Xa, factor XII, thrombin and plasmin do not hydrolyze B. bauhinioides inhibitor related substrates.

  13. Harms of Tumor Necrosis Factor Inhibitors in Rheumatic diseases: A focused Systematic Review of the Literature

    PubMed Central

    Jain, Archana; Singh, Jasvinder A.

    2013-01-01

    We performed a focused review of risk of harms of anti-tumor necrosis factor (TNF) inhibitors in adult rheumatic diseases. Increased risk of serious infections, tuberculosis and other opportunistic infections has been reported across various studies, with etanercept appearing to have modestly better safety profile in terms of tuberculosis and opportunistic infections and infliximab with higher risk of serious infections. Evidence suggests no increase in risk of cancer with anti-TNF biologics, but there is an increased risk of non-melanoma skin cancer. Elderly patients appear to be at increased risk of incident or worsening heart failure with anti-TNF biologic use. PMID:23444956

  14. Dermatologic Reactions to Targeted Therapy: A Focus on Epidermal Growth Factor Receptor Inhibitors and Nursing Care.

    PubMed

    Barton-Burke, Margaret; Ciccolini, Kathryn; Mekas, Maria; Burke, Sean

    2017-03-01

    Cancer treatments usually have side effects of bone marrow depression, mucositis, hair loss, and gastrointestinal issues. Rarely do we think of skin side effects until patients have been treated successfully with epidermal growth factor receptor inhibitors (EGFRi). Those reactions include papulopustular rash, hair changes, radiation dermatitis enhancement, pruritus, mucositis, xerosis, fissures, and paronychia. This article discusses the common skin reactions seen when using EGFRi and presents an overview of skin as the largest and important organ of the body, including an overview of skin assessment, pathophysiology of the skin reactions, nursing care involved, and introduction to oncodermatology.

  15. Factor VIII gene variants and inhibitor risk in African American hemophilia A patients.

    PubMed

    Gunasekera, Devi; Ettinger, Ruth A; Nakaya Fletcher, Shelley; James, Eddie A; Liu, Maochang; Barrett, John C; Withycombe, Janice; Matthews, Dana C; Epstein, Melinda S; Hughes, Richard J; Pratt, Kathleen P

    2015-08-13

    African American hemophilia A (HA) patients experience a higher incidence of neutralizing anti-factor VIII (FVIII) antibodies ("inhibitors") vis-à-vis white patients. Nonsynonymous single-nucleotide polymorphisms (ns-SNPs) in the F8 gene encoding FVIII-H484, FVIII-E1241, and FVIII-V2238 are more prevalent in African Americans. This study tested the hypothesis that immune responses to these sites provoke inhibitors. Blood samples were obtained from 174 African American and 198 white HA subjects and their F8 gene sequences determined. Major histocompatibility complex class II binding and T-cell recognition of polymorphic sequences were evaluated using quantitative binding assays and HLA-DRB1 tetramers. Peptides corresponding to 4 common ns-SNPs showed limited binding to 11 HLA-DRB1 proteins. CD4 T cells from 22 subjects treated with FVIII products having sequences at residues FVIII-484, 1241, and 2238 differing from those of putative proteins encoded by their F8 genes did not show high-avidity tetramer binding, whereas positive-control staining of tetanus-specific CD4 T cells was routinely successful. African Americans with an intron-22 inversion mutation showed a 2-3 times-higher inhibitor incidence than whites with the same mutation (odds ratio = 2.3 [1.1-5.0, P = .04]), but this did not correlate with any of the ns-SNPs. We conclude that immune responses to "sequence-mismatched" FVIII products are unlikely to contribute appreciably to the inhibitor incidence in African Americans.

  16. High-resolution genetic mapping of Xa27(t), a new bacterial blight resistance gene in rice, Oryza sativa L.

    PubMed

    Gu, K; Tian, D; Yang, F; Wu, L; Sreekala, C; Wang, D; Wang, G-L; Yin, Z

    2004-03-01

    Bacterial blight of rice, caused by Xanthomonas oryzae pv. oryzae ( Xoo) (Ishyama) Dye, is one of the serious diseases prevalent throughout Asia. In a previous study, a resistance ( R) locus was transferred from the tetraploid wild rice Oryza minuta to the cultivated rice species, Oryza sativa L. Here, we report the fine genetic mapping of the R locus, tentatively designated as Xa27(t). We performed disease evaluation with an Xa27(t) near-isogenic line, IRBB27, testing 35 Xoo strains collected from 11 countries. The Xa27(t) locus conferred a high level of resistance to 27 strains and moderate resistance to three strains. Resistance of the Xa27(t) gene was developmentally regulated in IRBB27 and showed semi-dominant or a dosage effect in the cv. CO39 genetic background. As a prelude to cloning Xa27(t), a molecular mapping strategy was employed with a large mapping population consisting of 3,875 gametes. Three molecular markers, M336, M1081, and M1059, closely linked to Xa27(t), were identified to facilitate the mapping of Xa27(t) to the long arm of chromosome 6. The Xa27(t) locus was confirmed by chromosome landing of M1081 and M1095 markers on the rice genome. Markers derived from the genomic sequence of O. sativa cv. Nipponbare were used to further saturate the Xa27(t) genomic region. Xa27(t) was finally located within a genetic interval of 0.052 cM, flanked by markers M964 and M1197, and co-segregated with markers M631, M1230, and M449.

  17. [Analysis of the Cochrane Review: Direct thrombin inhibitors versus vitamin K antagonists for preventing cerebral or systemic embolism in people with non-valvular atrial fibrillation. Cochrane Database Syst Rev. 2014,3:CD009893].

    PubMed

    Vaz Carneiro, António; Costa, João

    2014-01-01

    Ischemic stroke is one of the most important complications of lone (non-valvular) atrial fibrillation. Its prevention is usually accomplished through oral anticoagulation. Until a few years ago warfarin was the most used agent, but recently two new pharmacologic classes have been introduced for stroke prevention in these patients: oral direct thrombin inhibitors (dabigatran and ximelagatran) and oral factor Xa inhibitors (rivaroxaban, apixaban and edoxaban). In this systematic review, oral direct thrombin inhibitors were compared with warfarin for efficacy and safety. The results indicate that there is no difference in terms of efficacy (except dabigatran 150 mg BID). Oral direct thrombin inhibitors presented less hemorrhages but increased treatment withdrawal due to adverse side-effects (the authors performed post-hoc analyses excluding ximelagatran because this drug was withdrawn from the market owing to safety concerns). There was no difference in terms of mortality between the agents.

  18. Crystallization and preliminary crystallographic data for a ternary complex between tissue factor, factor VIIa and a BPTI-derived inhibitor

    NASA Astrophysics Data System (ADS)

    Stura, Enrico A.; Ruf, Wolfram; Wilson, Ian A.

    1996-10-01

    The binding of tissue factor (TF) with the serine protease coagulation factor VIIa (VIIa) is the initial trigger for activation of the coagulation protease cascades. In complex with TF, VIIa has profoundly enhanced function in the limited proteolytic activation of the natural substrate factors X and IX. Here we report the screening and identification of crystallization conditions to produce diffraction quality crystals of the complex between TF · VIIa and a potent inhibitor (5L 15) derived from mutagenesis of the bovine pancreatic trypsin inhibitor (BPTI) sequence. The complex crystals were obtained from the soluble extracellular domain of tissue factor, expressed in Escherichia coli as a fusion protein, VIIa expressed in mammalian cells and recombinant 5L15. Because only 1.5 mg of complex were available for this work, a reverse screening based strategy was used in the search and optimization of the crystallization conditions. Two different crystal forms were obtained from polyethylene glycol 4000 and monomethyl polyethylene glycol 2000 with cacodylate buffer at pH 6.5 in the presence of sodium and calcium ions. The addition of magnesium and zinc have profound effects on the crystallization. Both crystal forms are trigonal with cell parameters a = b = 129.3 Å, c = 110.8 Å and a = b = 67.2 Å, c = 314.8 Å diffracting to 7 and 3.2 Å resolution, respectively, each with one molecule in the asymmetric unit. Complete data sets have been collected from each of these forms to the resolution to which the crystals diffract. A structural understanding of the interaction of VIIa with its cofactor TF to form a binary enzyme, and its inhibition by 5L15 will provide a basis for the development of antithrombotic strategies.

  19. OsWRKY62 is a negative regulator of basal and Xa21-mediated defense against Xanthomonas oryzae pv. oryzae in rice

    USDA-ARS?s Scientific Manuscript database

    The rice Xa21 gene, which confers resistance to the bacterial pathogen Xanthomonas oryzae pv. oryzae (Xoo), encodes a receptor-like kinase. Few components involved in transducing the Xa21-mediated defense response have yet been identified. It is reported that XA21 binds to a WRKY transcription fac...

  20. Developing an Anti-Xa-Based Anticoagulation Protocol for Patients with Percutaneous Ventricular Assist Devices.

    PubMed

    Sieg, Adam; Mardis, B Andrew; Mardis, Caitlin R; Huber, Michelle R; New, James P; Meadows, Holly B; Cook, Jennifer L; Toole, J Matthew; Uber, Walter E

    2015-01-01

    Because of the complexities associated with anticoagulation in temporary percutaneous ventricular assist device (pVAD) recipients, a lack of standardization exists in their management. This retrospective analysis evaluates current anticoagulation practices at a single center with the aim of identifying an optimal anticoagulation strategy and protocol. Patients were divided into two cohorts based on pVAD implanted (CentriMag (Thoratec; Pleasanton, CA) / TandemHeart (CardiacAssist; Pittsburgh, PA) or Impella (Abiomed, Danvers, MA)), with each group individually analyzed for bleeding and thrombotic complications. Patients in the CentriMag/TandemHeart cohort were subdivided based on the anticoagulation monitoring strategy (activated partial thromboplastin time (aPTT) or antifactor Xa unfractionated heparin (anti-Xa) values). In the CentriMag/TandemHeart cohort, there were five patients with anticoagulation titrated based on anti-Xa values; one patient developed a device thrombosis and a major bleed, whereas another patient experienced major bleeding. Eight patients received an Impella pVAD. Seven total major bleeds in three patients and no thrombotic events were detected. Based on distinct differences between the devices, anti-Xa values, and outcomes, two protocols were created to guide anticoagulation adjustments. However, anticoagulation in patients who require pVAD support is complex with constantly evolving anticoagulation goals. The ideal level of anticoagulation should be individually determined using several coagulation laboratory parameters in concert with hemodynamic changes in the patient's clinical status, the device, and the device cannulation.

  1. Small molecule inhibitors of Late SV40 Factor (LSF) abrogate hepatocellular carcinoma (HCC): Evaluation using an endogenous HCC model

    PubMed Central

    Rajasekaran, Devaraja; Siddiq, Ayesha; Willoughby, Jennifer L.S.; Biagi, Jessica M.; Christadore, Lisa M.; Yunes, Sarah A.; Gredler, Rachel; Jariwala, Nidhi; Robertson, Chadia L.; Akiel, Maaged A.; Shen, Xue-Ning; Subler, Mark A.; Windle, Jolene J.; Schaus, Scott E.; Fisher, Paul B.; Hansen, Ulla; Sarkar, Devanand

    2015-01-01

    Hepatocellular carcinoma (HCC) is a lethal malignancy with high mortality and poor prognosis. Oncogenic transcription factor Late SV40 Factor (LSF) plays an important role in promoting HCC. A small molecule inhibitor of LSF, Factor Quinolinone Inhibitor 1 (FQI1), significantly inhibited human HCC xenografts in nude mice without harming normal cells. Here we evaluated the efficacy of FQI1 and another inhibitor, FQI2, in inhibiting endogenous hepatocarcinogenesis. HCC was induced in a transgenic mouse with hepatocyte-specific overexpression of c-myc (Alb/c-myc) by injecting N-nitrosodiethylamine (DEN) followed by FQI1 or FQI2 treatment after tumor development. LSF inhibitors markedly decreased tumor burden in Alb/c-myc mice with a corresponding decrease in proliferation and angiogenesis. Interestingly, in vitro treatment of human HCC cells with LSF inhibitors resulted in mitotic arrest with an accompanying increase in CyclinB1. Inhibition of CyclinB1 induction by Cycloheximide or CDK1 activity by Roscovitine significantly prevented FQI-induced mitotic arrest. A significant induction of apoptosis was also observed upon treatment with FQI. These effects of LSF inhibition, mitotic arrest and induction of apoptosis by FQI1s provide multiple avenues by which these inhibitors eliminate HCC cells. LSF inhibitors might be highly potent and effective therapeutics for HCC either alone or in combination with currently existing therapies. PMID:26313006

  2. Tissue factor pathway inhibitor reduces mortality from Escherichia coli septic shock.

    PubMed Central

    Creasey, A A; Chang, A C; Feigen, L; Wün, T C; Taylor, F B; Hinshaw, L B

    1993-01-01

    This study was designed to test the hypothesis that tissue factor pathway inhibitor (TFPI) plays a significant role in vivo in regulating coagulation that results from exposure of blood to tissue factor after vascular injury as in the case of gram negative sepsis. Highly purified recombinant TFPI (6 mg/kg) was administered either 30 min or 4 h after the start of a lethal intravenous Escherichia coli infusion in baboons. Early posttreatment of TFPI resulted in (a) permanent seven-day survivors (5/5) with significant improvement in quality of life, while the mean survival time for the controls (5/5) was 39.9 h (no survivors); and (b) significant attenuations of the coagulation response and various measures of cell injury, with significant reductions in pathology observed in E. coli sepsis target organs, including kidneys, adrenals, and lungs. TFPI administration did not affect the reduction in mean systemic arterial pressure, the increases in respiration and heart rate, or temperature changes associated with the bacterial infusion. TFPI treated E. coli infected baboons had significantly lower IL-6 levels than their phosphate buffered saline-treated controls, however tumor necrosis factor levels were similarly elevated in both groups. In contrast to the earlier 30-min treatment, the administration of TFPI at 4 h, i.e., 240 min, after the start of bacterial infusion resulted in prolongation of survival time, with 40% survival rate (2/5) and some attenuation of the coagulopathic response, especially in animals in which fibrinogen levels were above 10% of normal at the time of TFPI administration. Results provide evidence for the significance of tissue factor and tissue factor pathway inhibitor in bacterial sepsis, and suggest a role for blood coagulation in the regulation of the inflammatory response. PMID:8514893

  3. Discovery of Novel Human Epidermal Growth Factor Receptor-2 Inhibitors by Structure-based Virtual Screening

    PubMed Central

    Shi, Zheng; Yu, Tian; Sun, Rong; Wang, Shan; Chen, Xiao-Qian; Cheng, Li-Jia; Liu, Rong

    2016-01-01

    Background: Human epidermal growth factor receptor-2 (HER2) is a trans-membrane receptor like protein, and aberrant signaling of HER2 is implicated in many human cancers, such as ovarian cancer, gastric cancer, and prostate cancer, most notably breast cancer. Moreover, it has been in the spotlight in the recent years as a promising new target for therapy of breast cancer. Objective: Since virtual screening has become an integral part of the drug discovery process, it is of great significant to identify novel HER2 inhibitors by structure-based virtual screening. Materials and Methods: In this study, we carried out a series of elegant bioinformatics approaches, such as virtual screening and molecular dynamics (MD) simulations to identify HER2 inhibitors from Food and Drug Administration-approved small molecule drug as potential “new use” drugs. Results: Molecular docking identified top 10 potential drugs which showed spectrum affinity to HER2. Moreover, MD simulations suggested that ZINC08214629 (Nonoxynol-9) and ZINC03830276 (Benzonatate) might exert potential inhibitory effects against HER2-targeted anti-breast cancer therapeutics. Conclusion: Together, our findings may provide successful application of virtual screening studies in the lead discovery process, and suggest that our discovered small molecules could be effective HER2 inhibitor candidates for further study. SUMMARY A series of elegant bioinformatics approaches, including virtual screening and molecular dynamics (MD) simulations were took advantage to identify human epidermal growth factor receptor-2 (HER2) inhibitors. Molecular docking recognized top 10 candidate compounds, which showed spectrum affinity to HER2. Further, MD simulations suggested that ZINC08214629 (Nonoxynol-9) and ZINC03830276 (Benzonatate) in candidate compounds were identified as potential “new use” drugs against HER2-targeted anti-breast cancer therapeutics. Abbreviations used: HER2: Human epidermal growth factor receptor-2

  4. Therapeutic treatment with a novel hypoxia-inducible factor hydroxylase inhibitor (TRC160334) ameliorates murine colitis

    PubMed Central

    Gupta, Ram; Chaudhary, Anita R; Shah, Binita N; Jadhav, Avinash V; Zambad, Shitalkumar P; Gupta, Ramesh Chandra; Deshpande, Shailesh; Chauthaiwale, Vijay; Dutt, Chaitanya

    2014-01-01

    Background and aim Mucosal healing in inflammatory bowel disease (IBD) can be achieved by improvement of intestinal barrier protection. Activation of hypoxia-inducible factor (HIF) has been identified as a critical factor for barrier protection during mucosal insult and is linked with improvement in symptoms of colitis. Although prophylactic efficacy of HIF hydroxylase inhibitors in murine colitis have been established, its therapeutic efficacy in clinically relevant therapeutic settings have not been established. In the present study we aim to establish therapeutic efficacy of TRC160334, a novel HIF hydroxylase inhibitor, in animal models of colitis. Methods The efficacy of TRC160334 was evaluated in two different mouse models of colitis by oral route. A prophylactic efficacy study was performed in a 2,4,6-trinitrobenzene sulfonic acid-induced mouse model of colitis representing human Crohn’s disease pathology. Additionally, a therapeutic efficacy study was performed in a dextran sulfate sodium-induced mouse model of colitis, a model simulating human ulcerative colitis. Results TRC160334 treatment resulted in significant improvement in disease end points in both models of colitis. TRC160334 treatment resulted into cytoprotective heatshock protein 70 induction in inflamed colon. TRC160334 successfully attenuated the rate of fall in body weight, disease activity index, and macroscopic and microscopic scores of colonic damage leading to overall improvement in study outcome. Conclusion Our findings are the first to demonstrate that therapeutic intervention with a HIF hydroxylase inhibitor ameliorates IBD in disease models. These findings highlight the potential of TRC160334 for its clinical application in the treatment of IBD. PMID:24493931

  5. Natural Product-Based Inhibitors of Hypoxia-Inducible Factor-1 (HIF-1)

    PubMed Central

    Nagle, Dale G.; Zhou, Yu-Dong

    2010-01-01

    The transcription factor hypoxia-inducible factor-1 (HIF-1) regulates the expression of more than 70 genes involved in cellular adaptation and survival under hypoxic stress. Activation of HIF-1 is associated with numerous physiological and pathological processes that include tumorigenesis, vascular remodeling, inflammation, and hypoxia/ischemia-related tissue damage. Clinical studies suggested that HIF-1 activation correlates directly with advanced disease stages and treatment resistance among cancer patients. Preclinical studies support the inhibition of HIF-1 as a major molecular target for antitumor drug discovery. Considerable effort is underway, in government laboratories, industry and academia, to identify therapeutically useful small molecule HIF-1 inhibitors. Natural products (low molecular weight organic compounds produced by plants, microbes, and animals) continue to play a major role in modern antitumor drug discovery. Most of the compounds discovered to inhibit HIF-1 are natural products or synthetic compounds with structures that are based on natural product leads. Natural products have also served a vital role as molecular probes to elucidate the pathways that regulate HIF-1 activity. Natural products and natural product-derived compounds that inhibit HIF-1 are summarized in light of their biological source, chemical class, ancd effect on HIF-1 and HIF-mediated gene regulation. When known, the mechanism(s) of action of HIF-1 inhibitors are described. Many of the substances found to inhibit HIF-1 are non-druggable compounds that are too cytotoxic to serve as drug leads. The application of high-throughput screening methods, complementary molecular-targeted assays, and structurally diverse chemical libraries hold promise for the discovery of therapeutically useful HIF-1 inhibitors. PMID:16515532

  6. Sepsis-Induced Coagulation in the Baboon Lung Is Associated with Decreased Tissue Factor Pathway Inhibitor

    PubMed Central

    Tang, Haiwang; Ivanciu, Lacramioara; Popescu, Narcis; Peer, Glenn; Hack, Erik; Lupu, Cristina; Taylor, Fletcher B.; Lupu, Florea

    2007-01-01

    Increased tissue factor (TF)-dependent procoagulant activity in sepsis may be partly due to decreased expression or function of tissue factor pathway inhibitor (TFPI). To test this hypothesis, baboons were infused with live Escherichia coli and sacrificed after 2, 8, or 24 hours. Confocal and electron microscopy revealed increased leukocyte infiltration and fibrin deposition in the intravascular and interstitial compartments. Large amounts of TF were detected by immunostaining in leukocytes and platelet-rich microthrombi. TF induction was documented by quantitative reverse transcriptase-polymerase chain reaction, enzyme-linked immunosorbent assay, and coagulation assays. Lung-associated TFPI antigen and mRNA decreased during sepsis, and TFPI activity diminished abruptly at 2 hours. Blocking antibodies against TFPI increased fibrin deposition in septic baboon lungs, suggesting that TF-dependent coagulation might be aggravated by reduced endothelial TFPI. Decreased TFPI activity coincided with the release of tissue plasminogen activator and the peak of plasmin generation, suggesting that TFPI could undergo proteolytic inactivation by plasmin. Enhanced plasmin produced in septic baboons by infusion of blocking antibodies against plasminogen activator inhibitor-1 led to decreased lung-associated TFPI and unforeseen massive fibrin deposition. We conclude that activation of TF-driven coagulation not adequately countered by TFPI may underlie the widespread thrombotic complications of sepsis. PMID:17640967

  7. Inhibition of coagulation proteases Xa and IIa decreases ischemia-reperfusion injuries in a preclinical renal transplantation model.

    PubMed

    Tillet, Solenne; Giraud, Sébastien; Kerforne, Thomas; Saint-Yves, Thibaut; Joffrion, Sandrine; Goujon, Jean-Michel; Cau, Jerôme; Mauco, Gérard; Petitou, Maurice; Hauet, Thierry

    2016-12-01

    Coagulation is an important pathway in the pathophysiology of ischemia-reperfusion injuries. In particular, deceased after circulatory death (DCD) donors undergo a no-flow period, a strong activator of coagulation. Hence, therapies influencing the coagulation cascade must be developed. We evaluated the effect of a new highly specific and effective anti-Xa/IIa molecule, with an integrated innovative antidote site (EP217609), in a porcine preclinical model mimicking injuries observed in DCD donor kidney transplantation. Kidneys were clamped for 60 minutes (warm ischemia), then flushed and preserved for 24 hours at 4°C in University of Wisconsin (UW) solution (supplemented or not). EP217609-supplemented UW solution (UW-EP), compared with unfractionated heparin-supplemented UW solution (UW-UFH) or UW alone (UW). A mechanistic investigation was conducted in vitro: addition of EP217609 to endothelial cells during hypoxia at 4°C in the UW solution inhibited thrombin generation during reoxygenation at 37°C in human plasma and reduced tumor necrosis factor alpha, intercellular adhesion molecule 1, and vascular cell adhesion molecule 1 messenger RNA cell expressions. In vivo, function recovery was markedly improved in the UW-EP group. Interestingly, levels of thrombin-antithrombin complexes (reflecting thrombin generation) were reduced 60 minutes after reperfusion in the UW-EP group. In addition, 3 months after transplantation, lower fibrosis, epithelial-mesenchymal transition, inflammation, and leukocyte infiltration were observed. Using this new dual anticoagulant, anti-Xa/IIa activity during kidney flush and preservation is protected by reducing thrombin generation at revascularization, improving early function recovery, and decreasing chronic lesions. Such an easy-to-deploy clinical strategy could improve marginal graft outcome.

  8. Vascular endothelial growth factor (VEGF) and VEGF receptor inhibitors in the treatment of renal cell carcinomas.

    PubMed

    Roskoski, Robert

    2017-03-19

    One Von Hippel-Lindau (VHL) tumor suppressor gene is lost in most renal cell carcinomas while the nondeleted allele exhibits hypermethylation-induced inactivation or inactivating somatic mutations. As a result of these genetic modifications, there is an increased production of VEGF-A and pro-angiogenic growth factors in this disorder. The important role of angiogenesis in the pathogenesis of renal cell carcinomas and other tumors has focused the attention of investigators on the biology of VEGFs and VEGFR1-3 and to the development of inhibitors of the intricate and multifaceted angiogenic pathways. VEGFR1-3 contain an extracellular segment with seven immunoglobulin-like domains, a transmembrane segment, a juxtamembrane segment, a protein kinase domain with an insert of about 70 amino acid residues, and a C-terminal tail. VEGF-A stimulates the activation of preformed VEGFR2 dimers by the auto-phosphorylation of activation segment tyrosines followed by the phosphorylation of additional protein-tyrosines that recruit phosphotyrosine binding proteins thereby leading to signalling by the ERK1/2, AKT, Src, and p38 MAP kinase pathways. VEGFR1 modulates the activity of VEGFR2, which is the chief pathway in vasculogenesis and angiogenesis. VEGFR3 and its ligands (VEGF-C and VEGF-D) are involved primarily in lymphangiogenesis. Small molecule VEGFR1/2/3 inhibitors including axitinib, cabozantinib, lenvatinib, sorafenib, sunitinib, and pazopanib are approved by the FDA for the treatment of renal cell carcinomas. Most of these agents are type II inhibitors of VEGFR2 and inhibit the so-called DFG-Aspout inactive enzyme conformation. These drugs are steady-state competitive inhibitors with respect to ATP and like ATP they form hydrogen bonds with the hinge residues that connect the small and large protein kinase lobes. Bevacizumab, a monoclonal antibody that binds to VEGF-A, is also approved for the treatment of renal cell carcinomas. Resistance to these agents invariably occurs

  9. Nannocystin A: an Elongation Factor 1 Inhibitor from Myxobacteria with Differential Anti-Cancer Properties.

    PubMed

    Krastel, Philipp; Roggo, Silvio; Schirle, Markus; Ross, Nathan T; Perruccio, Francesca; Aspesi, Peter; Aust, Thomas; Buntin, Kathrin; Estoppey, David; Liechty, Brigitta; Mapa, Felipa; Memmert, Klaus; Miller, Howard; Pan, Xuewen; Riedl, Ralph; Thibaut, Christian; Thomas, Jason; Wagner, Trixie; Weber, Eric; Xie, Xiaobing; Schmitt, Esther K; Hoepfner, Dominic

    2015-08-24

    Cultivation of myxobacteria of the Nannocystis genus led to the isolation and structure elucidation of a class of novel cyclic lactone inhibitors of elongation factor 1. Whole genome sequence analysis and annotation enabled identification of the putative biosynthetic cluster and synthesis process. In biological assays the compounds displayed anti-fungal and cytotoxic activity. Combined genetic and proteomic approaches identified the eukaryotic translation elongation factor 1α (EF-1α) as the primary target for this compound class. Nannocystin A (1) displayed differential activity across various cancer cell lines and EEF1A1 expression levels appear to be the main differentiating factor. Biochemical and genetic evidence support an overlapping binding site of 1 with the anti-cancer compound didemnin B on EF-1α. This myxobacterial chemotype thus offers an interesting starting point for further investigations of the potential of therapeutics targeting elongation factor 1. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  10. Immune regulatory gene polymorphisms as predisposing risk factors for the development of factor VIII inhibitors in Indian severe haemophilia A patients.

    PubMed

    Pinto, P; Ghosh, K; Shetty, S

    2012-09-01

    Development of inhibitors to factor VIII, a serious complication of replacement therapy in haemophilia A patients, leads to increased bleeding, morbidity and mortality. There is no data on the risk factors for inhibitor development in Indian patients with severe haemophilia A. Our aim was to study the role of immune regulatory gene polymorphisms in the development of inhibitors. Fourteen immune regulatory gene polymorphisms (IL1β, IL4, IL10, TNFA and CTLA4) were analysed in 120 patients with severe haemophilia A, i.e. 50 inhibitor positive patients, and 70 inhibitor negative control patients, by PCR-RFLP, DNA sequencing and allele-specific PCRs. The IL10 promoter 'GCC' haplotypes overall (P: 0.002, OR: 3.452, 95% CI: 1.607-7.416), and 'GCC/ATA' (P: 0.011, OR: 3.492, 95% CI: 1.402-8.696) haplotype, associated with high and intermediate IL10 production, respectively, were significantly higher in inhibitor positive patients, whereas the 'non-GCC' haplotypes overall (P: 0.002,OR: 0.290, 95% CI 0.135-0.622) and 'ATA/ATA' haplotype (P: 0.025, OR: 0.278, 95% CI: 0.096-0.802), associated with low IL10 synthesis, were significantly higher among inhibitor negative patients. The TNFA rs1799724 C/T heterozygote prevalence was significantly higher in the inhibitor positive group (P: 0.021, OR: 3.190, 95% CI: 1.273-7.990), whereas the other polymorphisms showed no statistically significant association with the presence of inhibitors. Different immune regulatory gene polymorphisms play a significant role as possible risk factors for the development of inhibitors in severe haemophilia A patients.

  11. Missense mutations in the coagulation factor XII (Hageman factor) gene in hereditary angioedema with normal C1 inhibitor.

    PubMed

    Dewald, Georg; Bork, Konrad

    2006-05-19

    Hereditary angioedema is characterized by recurrent skin swelling, abdominal pain attacks, and potentially life-threatening upper airway obstruction. The two classic types are both caused by mutations within the complement C1 inhibitor gene. A recently described new type does not show a deficiency of C1 inhibitor and affects almost exclusively women. We screened twenty unrelated index patients with this new type of hereditary angioedema for mutations in the coagulation factor XII gene. Two different missense mutations were identified in exactly the same position within exon 9 of the F12 gene. 'Mutation 1' (1032C-->A), encountered in five patients, predicts a threonine-to-lysine substitution (Thr309Lys). 'Mutation 2' (1032C-->G), observed in one patient, results in a threonine-to-arginine substitution (Thr309Arg). The predicted structural and functional impact of the mutations, their absence in 145 healthy controls, and their co-segregation with the phenotype in five families provide strong support that they cause disease.

  12. Factor VIII alloantibody inhibitors: cost analysis of immune tolerance induction vs. prophylaxis and on-demand with bypass treatment.

    PubMed

    Earnshaw, S R; Graham, C N; McDade, C L; Spears, J B; Kessler, C M

    2015-05-01

    Development of inhibitors (alloantibodies to exogenous factor VIII) is the most significant treatment complication in patients with haemophilia A. The only proven way to eradicate inhibitors is through immune tolerance induction (ITI), while bypassing agents are typically employed to treat or prevent bleeds in patients with high titre inhibitors. Costs of these approaches have not been well studied. The aim of this study was to compare lifetime costs of treating patients with severe haemophilia A with inhibitors using on-demand or prophylaxis treatment with bypassing agents and ITI. A decision-analytic model was developed to compare the treatment costs and outcomes. Quantitation of the reduction in bleeding events for patients on prophylaxis and after eradication of inhibitors when on ITI and relapse of inhibitors was derived from published studies. Costs were obtained from standard US costing sources and are reported in 2014 US dollars. Costs and outcomes were discounted 3% per annum. Lifetime costs of treating patients with inhibitors are lower for ITI vs. on-demand or prophylaxis. Patients are also projected to live longer, have greater quality-adjusted life-years, and have fewer bleeding events than patients treated on-demand. Treating patients via ITI to eradicate inhibitors may result in lower lifetime costs and greater life-years and quality-adjusted life-years than treating with bypassing agents.

  13. Non-genetic risk factors in haemophilia A inhibitor management - the danger theory and the use of animal models.

    PubMed

    Lövgren, K M; Søndergaard, H; Skov, S; Wiinberg, B

    2016-09-01

    In haemophilia A (HA) management, antidrug antibodies, or inhibitors, are a serious complication that renders factor VIII (FVIII) replacement therapy ineffective, increases morbidity and reduces quality of life for affected patients. Inhibitor development aetiology is multifactorial and covers both genetic and therapy related risk factors. Many therapy-related risk factors have proven difficult to confirm due to several confounding factors and the small study populations available. However, clinical studies indicate that e.g. on-demand treatment and surgery affect inhibitor development, and explanations for this association are being investigated. A potential explanation is the danger signal effect, where the immune response is activated by endogenous or exogenous danger or damage signals present at the time and site of FVIII administration. The danger theory explains how alarm signals from stressed, injured or dying cells can activate an immune reaction, without the involvement of foreign antigens. Bleeds, trauma, surgery or concomitant infection could be events initiating danger signalling in HA patients, resulting in an immune reaction towards administered FVIII that otherwise would pass unnoticed. This role of danger in HA inhibitor formation has previously been suggested, but a thorough discussion of this subject is lacking. The present review will discuss the potential role of danger signals in haemophilia and inhibitor development, with focus on treatment related risk factors with a suspected danger signal aetiology; on-demand treatment, treatment during major bleeds or surgery, and treatment during infection or vaccination. Clinical studies as well as animal experiments addressing these factors will be reviewed.

  14. Thrombin inhibitors identified by computer-assisted multiparameter design

    PubMed Central

    Riester, Daniel; Wirsching, Frank; Salinas, Gabriela; Keller, Martina; Gebinoga, Michael; Kamphausen, Stefan; Merkwirth, Christian; Goetz, Ruediger; Wiesenfeldt, Martin; Stürzebecher, Jörg; Bode, Wolfram; Friedrich, Rainer; Thürk, Marcel; Schwienhorst, Andreas

    2005-01-01

    Here, we present a series of thrombin inhibitors that were generated by using powerful computer-assisted multiparameter optimization process. The process was organized in design cycles, starting with a set of randomly chosen molecules. Each cycle combined combinatorial synthesis, multiparameter characterization of compounds in a variety of bioassays, and algorithmic processing of the data to devise a set of compounds to be synthesized in the next cycle. The identified lead compounds exhibited thrombin inhibitory constants in the lower nanomolar range. They are by far the most selective synthetic thrombin inhibitors, with selectivities of >100,000-fold toward other proteases such as Factor Xa, Factor XIIa, urokinase, plasmin, and Plasma kallikrein. Furthermore, these compounds exhibit a favorable profile, comprising nontoxicity, high metabolic stability, low serum protein binding, good solubility, high anticoagulant activity, and a slow and exclusively renal elimination from the circulation in a rat model. Finally, x-ray crystallographic analysis of a thrombin–inhibitor complex revealed a binding mode with a neutral moiety in the S1 pocket of thrombin. PMID:15937115

  15. Cholesterol synthesis inhibitors protect against platelet-activating factor-induced neuronal damage

    PubMed Central

    Bate, Clive; Rumbold, Louis; Williams, Alun

    2007-01-01

    Background Platelet-activating factor (PAF) is implicated in the neuronal damage that accompanies ischemia, prion disease and Alzheimer's disease (AD). Since some epidemiological studies demonstrate that statins, drugs that reduce cholesterol synthesis, have a beneficial effect on mild AD, we examined the effects of two cholesterol synthesis inhibitors on neuronal responses to PAF. Methods Primary cortical neurons were treated with cholesterol synthesis inhibitors (simvastatin or squalestatin) prior to incubation with different neurotoxins. The effects of these drugs on neuronal cholesterol levels and neuronal survival were measured. Immunoblots were used to determine the effects of simvastatin or squalestatin on the distribution of the PAF receptor and an enzyme linked immunoassay was used to quantify the amounts of PAF receptor. Results PAF killed primary neurons in a dose-dependent manner. Pre-treatment with simvastatin or squalestatin reduced neuronal cholesterol and increased the survival of PAF-treated neurons. Neuronal survival was increased 50% by 100 nM simvastatin, or 20 nM squalestatin. The addition of mevalonate restored cholesterol levels, and reversed the protective effect of simvastatin. Simvastatin or squalestatin did not affect the amounts of the PAF receptor but did cause it to disperse from within lipid rafts. Conclusion Treatment of neurons with cholesterol synthesis inhibitors including simvastatin and squalestatin protected neurons against PAF. Treatment caused a percentage of the PAF receptors to disperse from cholesterol-sensitive domains. These results raise the possibility that the effects of statins on neurodegenerative disease are, at least in part, due to desensitisation of neurons to PAF. PMID:17233902

  16. Investigating the Regulation and Potential Role of Nonhypoxic Hypoxia Inducible Factor 1 (HIF 1) in Aromatase Inhibitor Resistant Breast Cancer

    DTIC Science & Technology

    2015-12-01

    ABSTRACT Prior to this study, aromatase inhibitor ( AI , ex. letrozole) resistance was associated withincreased dependence on growth factors and decreased...dependence on ERα. However, the role that such molecular changes play in AI resistance and the mechanism by which they elicit their effects were not...treatment of acquired AI resistance. 15. SUBJECT TERMS Breast cancer, aromatase inhibitors (ex. letrozole), drug resistance, cancer stem cells,nonhypoxic

  17. Inhibition of tissue factor pathway inhibitor increases the sensitivity of thrombin generation assay to procoagulant microvesicles.

    PubMed

    Gheldof, Damien; Mullier, François; Chatelain, Bernard; Dogné, Jean-Michel; Chatelain, Christian

    2013-07-01

    Patients with cancer have a seven-fold to 10-fold increased risk of developing venous thromboembolism (VTE). Circulating microvesicles could be a predictive biomarker for VTE in cancer. Thrombin generation assay (TGA) is a useful technique to detect procoagulant activity of microvesicles. However, TGA suffers from a lack of sensitivity due to the presence of tissue factor pathway inhibitor (TFPI) in plasma. The aim of the study was to improve the sensitivity of TGA to tissue factor by limiting the interference of TFPI. Serial dilutions of MDA-MB231 cells were incubated for 45 min at 37°C to generate microvesicles. Samples were then centrifuged and supernatants that contain microvesicles were used for TGA. Normal pooled plasma was incubated with inhibitor of TFPI or was diluted twice to decrease plasma level of TFPI. Lagtime was used as a surrogate marker of TGA to detect procoagulant activity of microvesicles. Inhibition of TFPI decreased twice the cell concentration needed for a significant reduction of lagtime and decreased 2.4-fold the intraassay variability. Plasma dilution had no impact on the TGA sensitivity when TGA was triggered by microvesicles derived from MDA-MB-231. Thrombin generation is a very sensitive method to study the procoagulant activity of tissue factor bearing microvesicles. The sensitivity can be increased by inhibition of TFPI with specific monoclonal antibody against its Kunitz domain I. A two times plasma dilution is an interesting cheaper alternative to study the procoagulant activity of microvesicles by TGA with a good sensitivity, especially when low plasma quantities are available.

  18. Coexpression of heparanase activity, cathepsin L, tissue factor, tissue factor pathway inhibitor, and MMP-9 in proliferative diabetic retinopathy

    PubMed Central

    Siddiquei, Mohammad Mairaj; Nawaz, Mohd Imtiaz; De Hertogh, Gert; Mohammad, Ghulam; Alam, Kaiser; Mousa, Ahmed; Opdenakker, Ghislain

    2016-01-01

    Purpose Heparanase cleaves heparan sulfate side chains of heparan sulfate proteoglycans, activity that is implicated in angiogenesis. Proteolytic cleavage of proheparanase by cathepsin L leads to the formation of catalytically active heparanase. We investigated the expression levels of heparanase enzymatic activity and correlated these with the levels of cathepsin L, the angiogenic factors tissue factor (TF) and matrix metalloproteinase-9 (MMP-9), and the angiostatic factor tissue factor pathway inhibitor (TFPI) in proliferative diabetic retinopathy (PDR). Methods Vitreous samples from 25 patients with PDR and 20 nondiabetic patients and epiretinal membranes from 12 patients with PDR were studied with enzyme-linked immunosorbent assay, western blot analysis, and immunohistochemistry. Results We observed a significant increase in the expression of heparanase activity in vitreous samples from patients with PDR compared to the nondiabetic controls (p=0.027). Significant positive correlations were found between the levels of heparanase activity and the levels of cathepsin L (r=0.51; p=0.001), TF (r=0.6; p<0.0001), and TFPI (r=0.49; p=0.001). The expression levels of cathepsin L (p=0.019), TF (p<0.0001), TFPI (p<0.0001), and MMP-9 (p=0.029) were significantly higher in the vitreous samples with detected heparanase activity compared to the vitreous samples with undetected heparanase activity. Western blot analysis demonstrated proteolytic cleavage of TFPI in the vitreous samples from patients with PDR. In the epiretinal membranes, cathepsin L, TF, and TFPI were expressed in vascular endothelial cells and CD45-expressing leukocytes. Significant positive correlations were detected between the number of blood vessels that expressed CD31 and the number of blood vessels that expressed TF (r=0.9; p<0.0001) and TFPI (r=0.81; p=0.001). Conclusions The coexpression of these angiogenesis regulatory factors suggests cross-talk between these factors and pathogenesis of PDR

  19. A Highly Selective Dual Insulin Receptor (IR)/Insulin-like Growth Factor 1 Receptor (IGF-1R) Inhibitor Derived from an Extracellular Signal-regulated Kinase (ERK) Inhibitor*

    PubMed Central

    Anastassiadis, Theonie; Duong-Ly, Krisna C.; Deacon, Sean W.; Lafontant, Alec; Ma, Haiching; Devarajan, Karthik; Dunbrack, Roland L.; Wu, Jinhua; Peterson, Jeffrey R.

    2013-01-01

    Dual inhibitors of the closely related receptor tyrosine kinases insulin-like growth factor 1 receptor (IGF-1R) and insulin receptor (IR) are promising therapeutic agents in cancer. Here, we report an unusually selective class of dual inhibitors of IGF-1R and IR identified in a parallel screen of known kinase inhibitors against a panel of 300 human protein kinases. Biochemical and structural studies indicate that this class achieves its high selectivity by binding to the ATP-binding pocket of inactive, unphosphorylated IGF-1R/IR and stabilizing the activation loop in a native-like inactive conformation. One member of this compound family was originally reported as an inhibitor of the serine/threonine kinase ERK, a kinase that is distinct in the structure of its unphosphorylated/inactive form from IR/IGF-1R. Remarkably, this compound binds to the ATP-binding pocket of ERK in an entirely different conformation to that of IGF-1R/IR, explaining the potency against these two structurally distinct kinase families. These findings suggest a novel approach to polypharmacology in which two or more unrelated kinases are inhibited by a single compound that targets different conformations of each target kinase. PMID:23935097

  20. Discovery of Novel Fibroblast Growth Factor Receptor 1 Kinase Inhibitors by Structure-Based Virtual Screening

    SciTech Connect

    Ravindranathan, K.; Mandiyan, V; Ekkati, A; Bae, J; Schlessinger, J; Jorgensen, W

    2010-01-01

    Fibroblast growth factors (FGFs) play important roles in embryonic development, angiogenesis, wound healing, and cell proliferation and differentiation. In search of inhibitors of FGFR1 kinase, 2.2 million compounds were docked into the ATP binding site of the protein. A co-crystal structure, which shows two alternative conformations for the nucleotide binding loop, is reported. Docking was performed on both conformations and, ultimately, 23 diverse compounds were purchased and assayed. Following hit validation, two compounds 10 and 16, a benzylidene derivative of pseudothiohydantoin and a thienopyrimidinone derivative, respectively, were discovered that inhibit FGFR1 kinase with IC{sub 50} values of 23 and 50 {micro}M. Initial optimization of 16 led to the more unsaturated 40, which has significantly enhanced potency, 1.9 {micro}M. The core structures represent new structural motifs for FGFR1 kinase inhibitors. The study also illustrates complexities associated with the choice of protein structures for docking, possible use of multiple kinase structures to seek selectivity, and hit identification.

  1. Treatment with tumour necrosis factor inhibitor oxpentifylline does not improve corticosteroid dependent chronic active Crohn's disease.

    PubMed Central

    Bauditz, J; Haemling, J; Ortner, M; Lochs, H; Raedler, A; Schreiber, S

    1997-01-01

    BACKGROUND: In Crohn's disease, inflammation is presumably sustained by an increased production of proinflammatory cytokines, in particular tumour necrosis factor alpha (TNF alpha) and interleukin 1 beta (IL 1 beta). TNF alpha can induce a host of cellular effector events resulting in perpetuation of the inflammatory process. In vivo studies with anti-TNF alpha antibody treatment have led to impressive clinical results. AIMS: To investigate whether treatment with the TNF alpha inhibitor oxpentifylline results in clinical improvement in corticosteroid dependent chronic active Crohn's disease. METHODS: Sixteen Crohn's disease patients received oxpentifylline 400 mg four times a day in a four week open label study. RESULTS: Blockade of TNF alpha production in 16 patients with corticosteroid dependent Crohn's disease did not improve the clinical disease activity (CDAI mean (SEM) 188.75 (5.65) versus 185.13 (10.87) or the endoscopic degree of inflammation (CDEIS 14.9 (2.87) versus 14.8 (2.27) or laboratory parameters. CONCLUSIONS: In this study, use of the TNF alpha inhibitor oxpentifylline does not improve inflammation in Crohn's disease. This finding suggests that there may be more key mediators than only TNF alpha in the inflammatory process in Crohn's disease. PMID:9176073

  2. Interactions of ABCG2 (BCRP) with epidermal growth factor receptor kinase inhibitors developed for molecular imaging.

    PubMed

    Qawasmi, Israa; Shmuel, Miriam; Eyal, Sara

    2014-01-01

    The objective of this study was to investigate in vitro the interactions between novel epidermal growth factor receptor kinase inhibitors (EGFRIs) developed for positron emission tomography (PET) imaging and the major efflux transporter breast cancer resistance protein (BCRP/ABCG2). Seven compounds were evaluated, using the ATPase activity assays and Madin-Darbey canine kidney (MDCK) cells overexpressing BCRP. Five of the tested compounds activated BCRP ATPase to various extent. Overexpression of BCRP conferred resistance to ML04, ML06, methoxy-Br-ML03, and PEG6-ML05 (IC50 values for inhibition of control cell proliferation 2.1 ± 0.6, 2.2 ± 0.7, 1.8 ± 1.2, and 2.8 ± 3.1 μM, respectively, compared to >50 μM in MDCK-BCRP cells). At submicromolar concentrations, none of the EGFRIs significantly inhibited BCRP. Immunoblotting studies indicated that BCRP expression is evident in cell lines utilized for in vivo tumor grafting in small animal PET imaging studies. Thus, the intensity of EGFRIs radioactivity signals previously observed in tumor xenografts reflects an interplay between transporter-mediated distribution of the probe into tumor cells and target binding. Concomitant use of efflux transporter inhibitors may help distinguish between the contribution of efflux transport and EGFR binding to the tissue signal.

  3. Interactions of ABCG2 (BCRP) with epidermal growth factor receptor kinase inhibitors developed for molecular imaging

    PubMed Central

    Qawasmi, Israa; Shmuel, Miriam; Eyal, Sara

    2014-01-01

    The objective of this study was to investigate in vitro the interactions between novel epidermal growth factor receptor kinase inhibitors (EGFRIs) developed for positron emission tomography (PET) imaging and the major efflux transporter breast cancer resistance protein (BCRP/ABCG2). Seven compounds were evaluated, using the ATPase activity assays and Madin-Darbey canine kidney (MDCK) cells overexpressing BCRP. Five of the tested compounds activated BCRP ATPase to various extent. Overexpression of BCRP conferred resistance to ML04, ML06, methoxy-Br-ML03, and PEG6-ML05 (IC50 values for inhibition of control cell proliferation 2.1 ± 0.6, 2.2 ± 0.7, 1.8 ± 1.2, and 2.8 ± 3.1 μM, respectively, compared to >50 μM in MDCK-BCRP cells). At submicromolar concentrations, none of the EGFRIs significantly inhibited BCRP. Immunoblotting studies indicated that BCRP expression is evident in cell lines utilized for in vivo tumor grafting in small animal PET imaging studies. Thus, the intensity of EGFRIs radioactivity signals previously observed in tumor xenografts reflects an interplay between transporter-mediated distribution of the probe into tumor cells and target binding. Concomitant use of efflux transporter inhibitors may help distinguish between the contribution of efflux transport and EGFR binding to the tissue signal. PMID:25484865

  4. KRAS mutational status as a predictor of epidermal growth factor receptor inhibitor efficacy in colorectal cancer.

    PubMed

    Baynes, Roy D; Gansert, Jennifer

    2009-01-01

    Inhibitors of the epidermal growth factor receptor (EGFR) have demonstrated promising potential in the treatment of advanced colorectal cancer. However, a proportion of patients do not respond to therapy with EGFR inhibitors, and therefore, there has been interest in identifying those patients most likely to benefit from therapy with these agents. KRAS, a member of the RAS family of signaling proteins, plays an important role in EGFR-mediated regulation of cellular proliferation and survival. Although there is still some debate regarding the prognostic importance of KRAS mutations in patients with metastatic colorectal cancer, several recent phase 2 and 3 studies have identified the presence of mutations at codons 12 and 13 of KRAS as predictors of poor response to the anti-EGFR monoclonal antibodies panitumumab and cetuximab. Patients with wild-type KRAS were found to have significantly better progression-free survival, overall survival, and/or objective response rate compared with patients harboring KRAS mutations. As a result, there has been growing interest in the development of KRAS mutational status as a biomarker for predicting patient response to EGFR-targeted therapy. Screening colorectal tumors for the absence of KRAS mutations may help identify patients most likely to benefit from anti-EGFR therapies.

  5. Ethnic differences in response to epidermal growth factor receptor tyrosine kinase inhibitors.

    PubMed

    Calvo, Emiliano; Baselga, José

    2006-05-10

    The identification of somatic mutations in the tyrosine kinase domain of the epidermal growth factor receptor (EGFR) in non-small-cell lung cancer (NSCLC) and their correlation with response to EGFR inhibitors has become an important event in the fields of cancer genetics and therapeutics. The initial observation of a higher response to gefitinib and erlotinib in patients of Asian origin was followed by the discovery that they harbor more frequent EGFR mutations in NSCLC; this raises the issue of ethnic diversity in the pathogenesis of given tumors. In a similar fashion, amplification of the closely related HER2 gene, which could also have implications for the treatment of NSCLC, is also more frequent in East Asian patients. On the other hand, EGFR gene amplification may be more prevalent in Western populations. The implication of these findings is that ethnicity may indicate different genetic backgrounds in common tumors that may influence clinical outcome and response to therapy. Therefore, in clinical trials with tyrosine kinase inhibitors and other molecular-targeted therapies, the inclusion of a global population appears to be required.

  6. The vascular endothelial growth factor receptor inhibitor PTK787/ZK222584 inhibits aromatase.

    PubMed

    Banerjee, Susana; Zvelebil, Marketa; Furet, Pascal; Mueller-Vieira, Ursula; Evans, Dean B; Dowsett, Mitch; Martin, Lesley-Ann

    2009-06-01

    Endocrine therapy is well established for the treatment of breast cancer, and antiangiogenic agents are showing considerable promise. Targeting the vascular endothelial growth factor (VEGF) and estrogen receptor (ER) signaling pathways concomitantly may provide enhanced therapeutic benefit in ER-positive breast cancer. Therefore, the effects of the VEGF receptor (VEGFR) tyrosine kinase inhibitor PTK787/ZK222584 (PTK/ZK) were investigated using human breast cancer cell lines engineered to express aromatase. As expected in this system, estrogen (E2) or androstenedione induced a proliferative response and increased ER-mediated transcription in ER-positive cell lines expressing aromatase. However, surprisingly, in the presence of androstenedione, PTK/ZK suppressed both the androstenedione-stimulated proliferation and ER-mediated transcription. PTK/ZK alone and in the presence of E2 had no observable effect on proliferation or ER-mediated transcription. These effects result from PTK/ZK having previously unrecognized antiaromatase activity and PTK/ZK being a competitive aromatase inhibitor. Computer-assisted molecular modeling showed that PTK/ZK could potentially bind directly to aromatase. The demonstration that PTK/ZK inhibits aromatase and VEGFR indicates that agents cross-inhibiting two important classes of targets in breast cancer could be developed.

  7. Resistance to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-Small Cell Lung Cancer.

    PubMed

    Hammerman, Peter S; Jänne, Pasi A; Johnson, Bruce E

    2009-12-15

    Gefitinib and erlotinib are ATP competitive inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase and are approved around the world for the treatment of patients with non-small cell lung cancer (NSCLC). Somatic mutations in the EGFR are found in 10 to 40% of patients with NSCLC. Patients with sensitizing somatic mutations of EGFR treated with gefitinib or erlotinib have an initial clinical response of 60 to 80%, approximately twice as high as the responses associated with the administration of conventional platinum-based chemotherapy. However, the efficacy of EGFR tyrosine kinase inhibitors (TKI) is limited by either primary (de novo) or acquired resistance after therapy and investigations to define the mechanisms of resistance are active areas of ongoing preclinical and clinical studies. Primary resistance is typically caused by other somatic mutations in genes such as KRAS, which also have an impact on the EGFR signaling pathway or by mutations in the EGFR gene that are not associated with sensitivity to EGFR-TKIs. Two established mechanisms of acquired resistance are caused by additional mutations in the EGFR gene acquired during the course of treatment that change the protein-coding sequence or by amplification of another oncogene signaling pathway driven by the MET oncogene. This review focuses on characterized mechanisms of resistance to the EGFR TKIs and efforts to overcome the problem of resistance aimed at improving the therapy of patients with NSCLC. (Clin Cancer Res 2009;15(24):7502-9).

  8. Finding of the Low Molecular Weight Inhibitors of Resuscitation Promoting Factor Enzymatic and Resuscitation Activity

    PubMed Central

    Demina, Galina R.; Makarov, Vadim A.; Nikitushkin, Vadim D.; Ryabova, Olga B.; Vostroknutova, Galina N.; Salina, Elena G.; Shleeva, Margarita O.; Goncharenko, Anna V.; Kaprelyants, Arseny S.

    2009-01-01

    Background Resuscitation promoting factors (RPF) are secreted proteins involved in reactivation of dormant actinobacteria, including Mycobacterium tuberculosis. They have been considered as prospective targets for the development of new anti-tuberculosis drugs preventing reactivation of dormant tubercle bacilli, generally associated with latent tuberculosis. However, no inhibitors of Rpf activity have been reported so far. The goal of this study was to find low molecular weight compounds inhibiting the enzymatic and biological activities of Rpfs. Methodology/Principal Findings Here we describe a novel class of 2-nitrophenylthiocyanates (NPT) compounds that inhibit muralytic activity of Rpfs with IC50 1–7 µg/ml. Fluorescence studies revealed interaction of active NPTs with the internal regions of the Rpf molecule. Candidate inhibitors of Rpf enzymatic activity showed a bacteriostatic effect on growth of Micrococcus luteus (in which Rpf is essential for growth protein) at concentrations close to IC50. The candidate compounds suppressed resuscitation of dormant (“non-culturable”) cells of M. smegmatis at 1 µg/ml or delayed resuscitation of dormant M. tuberculosis obtained in laboratory conditions at 10 µg/ml. However, they did not inhibit growth of active mycobacteria under these concentrations. Conclusions/Significance NPT are the first example of low molecular weight compounds that inhibit the enzymatic and biological activities of Rpf proteins. PMID:20016836

  9. Cutaneous toxicity from epidermal growth factor receptor inhibitors: would a subcutaneous desensitization be helpful? Case report.

    PubMed

    D'Alessio, Andrea; Cecchini, Sara; Di Mauro, Daniela; Geroli, Luca; Villa, Simonetta; Quadri, Antonello; Resta, Davide; Fortugno, Carmelo

    2016-11-11

    Cetuximab and panitumumab are monoclonal antibody inhibitors that bind the epidermal growth factor receptor (EGFR) currently used in the treatment of metastatic colorectal cancer. The main adverse event related to EGFR inhibitors (EGFR-Is) is cutaneous toxicity, which can cause dosage reduction and interruption of treatment. State-of-the-art management of skin toxicity associated with EGFR-Is therapy involves the topical administration of corticosteroids and oral antibiotics, but is not completely effective in the management of toxicity. Subcutaneous desensitization with increasing concentrations of monoclonal antibodies can induce a tolerance to drug administration and reduce cutaneous adverse effects. To our knowledge, this is the first case in which a reduction or a disappearance of skin toxicity caused by EGFR-Is through subcutaneous desensitization has been achieved. We present cases of 2 Caucasian patients with adenocarcinoma of the colon treated with EGFR-Is who developed severe cutaneous toxicity. A 73-year-old man presented grade 4 skin toxicity of the face and grade 3 skin toxicity of the trunk during treatment with cetuximab. A 68-year-old woman developed G2 rash on the face after the first administration of cetuximab. These patients underwent subcutaneous desensitization with increasing concentrations of EGFR-Is. After this procedure, patients restarted therapy at the optimal dosage with reduction or disappearance of skin toxicity. These cases suggest that by giving rising doses of antibody it is possible to obtain desensitization able to prevent severe cutaneous adverse events in patients treated with EGFR-Is.

  10. Management of adverse events during treatment of gastrointestinal cancers with epidermal growth factor inhibitors.

    PubMed

    Hofheinz, Ralf-Dieter; Segaert, Siegfried; Safont, María José; Demonty, Gaston; Prenen, Hans

    2017-06-01

    The epidermal growth factor receptor (EGFR) is involved in development and progression of some gastrointestinal cancers, and is targeted by monoclonal antibodies (mAbs) and tyrosine kinase inhibitors (TKIs) used to treat these conditions. Targeted agents are generally better tolerated than conventional chemotherapy, but have characteristic toxicities that can affect adherence, dosing, and outcomes. Skin conditions are the most common toxicities associated with EGFR inhibitors, particularly papulopustular rash. Other common toxicities include mucosal toxicity, electrolyte imbalances (notably hypomagnesaemia), and diarrhoea, while the chimaeric mAb cetuximab is also associated with increased risk of infusion reactions. With appropriate prophylaxis, the incidence and severity of these events can be reduced, while management strategies tailored to the patient and the degree of toxicity can help to ensure continuation of anti-cancer therapy. Here, we review the main toxicities associated with EGFR-inhibiting mAbs and TKIs in patients with gastrointestinal cancers, and provide recommendations for prophylaxis and treatment. Copyright © 2017 The Authors. Published by Elsevier B.V. All rights reserved.

  11. Mutational activation of BRAF confers sensitivity to transforming growth factor beta inhibitors in human cancer cells

    PubMed Central

    Spender, Lindsay C.; Ferguson, G. John; Liu, Sijia; Cui, Chao; Girotti, Maria Romina; Sibbet, Gary; Higgs, Ellen B.; Shuttleworth, Morven K.; Hamilton, Tom; Lorigan, Paul; Weller, Michael; Vincent, David F.; Sansom, Owen J.; Frame, Margaret; Dijke, Peter ten; Marais, Richard; Inman, Gareth J.

    2016-01-01

    Recent data implicate elevated transforming growth factor-β (TGFβ) signalling in BRAF inhibitor drug-resistance mechanisms, but the potential for targeting TGFβ signalling in cases of advanced melanoma has not been investigated. We show that mutant BRAFV600E confers an intrinsic dependence on TGFβ/TGFβ receptor 1 (TGFBR1) signalling for clonogenicity of murine melanocytes. Pharmacological inhibition of the TGFBR1 blocked the clonogenicity of human mutant BRAF melanoma cells through SMAD4-independent inhibition of mitosis, and also inhibited metastasis in xenografted zebrafish. When investigating the therapeutic potential of combining inhibitors of mutant BRAF and TGFBR1, we noted that unexpectedly, low-dose PLX-4720 (a vemurafenib analogue) promoted proliferation of drug-naïve melanoma cells. Pharmacological or pharmacogenetic inhibition of TGFBR1 blocked growth promotion and phosphorylation of SRC, which is frequently associated with vemurafenib-resistance mechanisms. Importantly, vemurafenib-resistant patient derived cells retained sensitivity to TGFBR1 inhibition, suggesting that TGFBR1 could be targeted therapeutically to combat the development of vemurafenib drug-resistance. PMID:27835901

  12. Brunner's gland lesions in rats induced by a vascular endothelial growth factor receptor inhibitor.

    PubMed

    Inomata, Akira; Nakano-Ito, Kyoko; Fujikawa, Yasuhiro; Sonoda, Jiro; Hayakawa, Kazuhiro; Ohta, Etsuko; Taketa, Yoshikazu; Van Gessel, Yvonne; Akare, Sandeep; Hutto, David; Hosokawa, Satoru; Tsukidate, Kazuo

    2014-12-01

    Vascular endothelial growth factor (VEGF) receptor tyrosine kinase (RTK) inhibitors are reported to cause reversible mucosal hyperplasia (adenosis) in the duodenum of rats; however, the pathogenesis is not fully elucidated. Using lenvatinib, a VEGF RTK inhibitor, we characterized the histologic time course of this duodenal change in rats. At 4 weeks, there was degeneration and necrosis of Brunner's gland epithelium accompanied by neutrophil infiltration around the affected glands. At 13 weeks, the inflammation was more extensive, and Brunner's gland epithelium was attenuated and flattened and was accompanied by reactive hyperplasia of duodenal epithelium. At 26 weeks, the changes became more severe and chronic and characterized by marked cystic dilation, which extended to the external muscular layer. These dilated glands exhibited morphological characteristics of duodenal crypt epithelium, suggestive of replacement of disappeared Brunner's glands by regenerative duodenal crypt epithelial cells. Similar changes were not present in similar time course studies in dog and monkey studies, suggesting that this is a rodent- or species-specific change. Based on the temporal progression of Brunner's gland lesion, we identify degeneration and necrosis of the Brunner's glands as the primary change leading to inflammation, cystic dilatation, and regeneration with cells that are morphologically suggestive of duodenal crypt epithelium.

  13. The Rice TAL Effector–Dependent Resistance Protein XA10 Triggers Cell Death and Calcium Depletion in the Endoplasmic Reticulum[W

    PubMed Central

    Tian, Dongsheng; Wang, Junxia; Zeng, Xuan; Gu, Keyu; Qiu, Chengxiang; Yang, Xiaobei; Zhou, Zhiyun; Goh, Meiling; Luo, Yanchang; Murata-Hori, Maki; White, Frank F.; Yin, Zhongchao

    2014-01-01

    The recognition between disease resistance (R) genes in plants and their cognate avirulence (Avr) genes in pathogens can produce a hypersensitive response of localized programmed cell death. However, our knowledge of the early signaling events of the R gene–mediated hypersensitive response in plants remains limited. Here, we report the cloning and characterization of Xa10, a transcription activator–like (TAL) effector-dependent R gene for resistance to bacterial blight in rice (Oryza sativa). Xa10 contains a binding element for the TAL effector AvrXa10 (EBEAvrXa10) in its promoter, and AvrXa10 specifically induces Xa10 expression. Expression of Xa10 induces programmed cell death in rice, Nicotiana benthamiana, and mammalian HeLa cells. The Xa10 gene product XA10 localizes as hexamers in the endoplasmic reticulum (ER) and is associated with ER Ca2+ depletion in plant and HeLa cells. XA10 variants that abolish programmed cell death and ER Ca2+ depletion in N. benthamiana and HeLa cells also abolish disease resistance in rice. We propose that XA10 is an inducible, intrinsic terminator protein that triggers programmed cell death by a conserved mechanism involving disruption of the ER and cellular Ca2+ homeostasis. PMID:24488961

  14. Cost of dose escalation in people with rheumatoid arthritis treated with tumour necrosis factor inhibitors across Europe.

    PubMed

    Holden, Sarah E; Currie, Craig J; Lennon, Martin; Reynolds, Alan V; Moots, Robert J

    2016-01-01

    The aim of this study was to calculate the marginal cost of dose escalation in people with rheumatoid arthritis treated with tumour necrosis factor (TNF) inhibitors across Europe. The proportion of people who escalate their dose of TNF inhibitor and the average percentage increase in TNF inhibitor cost associated with escalators versus non-escalators was calculated from previously published estimates, weighted by the sample size for each study. The number of people with rheumatoid arthritis treated with TNF inhibitors and the corresponding total drug sales were obtained for five European countries from Decision Resources' Pharmacor Market Forecast. Method 1 assumed that total sales of a TNF inhibitor represented the cost of an escalator multiplied by the number of escalators plus the cost of a non-escalator multiplied by the number of non-escalators. Method 2 assumed that the drug cost per day used to forecast total sales was calculated using the dose of TNF inhibitor used by non-escalators. The marginal cost of TNF inhibitor dose escalation was estimated by multiplying the difference in cost between escalators and non-escalators by the number of escalators. The estimated increase in TNF inhibitor costs associated with dose escalation in people with rheumatoid arthritis across five European countries (Germany, France, UK, Spain and Italy) was €51.5-54.4 million for adalimumab, €44.8-52.8 million for infliximab and €5.8-5.9 million for etanercept. Dose escalation of the TNF inhibitors adalimumab, etanercept and infliximab in people with rheumatoid arthritis has resulted in an increase in TNF inhibitor costs across five European countries.

  15. Synthesis and Structure-activity Analysis of Diphenylpyrazolodiazene Inhibitors of the HIV-1 Nef Virulence Factor

    PubMed Central

    Iyer, Prema C.; Zhao, Jielu; Emert-Sedlak, Lori A.; Moore, Kerry; Smithgall, Thomas E.; Day, Billy W.

    2014-01-01

    HIV-1 Nef is a critical AIDS progression factor yet underexplored target for antiretroviral drug discovery. A recent high-throughput screen for pharmacological inhibitors of Nef-dependent Src-family kinase activation identified a diphenylpyrazolodiazene hit compound with submicromolar potency in HIV-1 replication assays against a broad range of primary Nef variants. This compound, known as ‘B9’, binds directly to Nef and inhibits is dimerization in cells as a possible mechanism of action. Here were synthesized a diverse set of B9 analogs and identified structural features essential to antiretroviral activity. Chemical modifications to each of the three rings present in the parent compound were identified that did not compromise antiviral action. These analogs will guide the development of next-generation compounds with appropriate pharmacological profiles for assessment of antiretroviral activity in vivo. PMID:24650642

  16. QSAR and 3D QSAR of inhibitors of the epidermal growth factor receptor

    NASA Astrophysics Data System (ADS)

    Pinto-Bazurco, Mariano; Tsakovska, Ivanka; Pajeva, Ilza

    This article reports quantitative structure-activity relationships (QSAR) and 3D QSAR models of 134 structurally diverse inhibitors of the epidermal growth factor receptor (EGFR) tyrosine kinase. Free-Wilson analysis was used to derive the QSAR model. It identified the substituents in aniline, the polycyclic system, and the substituents at the 6- and 7-positions of the polycyclic system as the most important structural features. Comparative molecular field analysis (CoMFA) and comparative molecular similarity indices analysis (CoMSIA) were used in the 3D QSAR modeling. The steric and electrostatic interactions proved the most important for the inhibitory effect. Both QSAR and 3D QSAR models led to consistent results. On the basis of the statistically significant models, new structures were proposed and their inhibitory activities were predicted.

  17. Delirium in the geriatric unit: proton-pump inhibitors and other risk factors

    PubMed Central

    Otremba, Iwona; Wilczyński, Krzysztof; Szewieczek, Jan

    2016-01-01

    Background Delirium remains a major nosocomial complication of hospitalized elderly. Predictive models for delirium may be useful for identification of high-risk patients for implementation of preventive strategies. Objective Evaluate specific factors for development of delirium in a geriatric ward setting. Methods Prospective cross-sectional study comprised 675 consecutive patients aged 79.2±7.7 years (66% women and 34% men), admitted to the subacute geriatric ward of a multiprofile university hospital after exclusion of 113 patients treated with antipsychotic medication because of behavioral disorders before admission. Comprehensive geriatric assessments including a structured interview, physical examination, geriatric functional assessment, blood sampling, ECG, abdominal ultrasound, chest X-ray, Confusion Assessment Method for diagnosis of delirium, Delirium-O-Meter to assess delirium severity, Richmond Agitation-Sedation Scale to assess sedation or agitation, visual analog scale and Doloplus-2 scale to assess pain level were performed. Results Multivariate logistic regression analysis revealed five independent factors associated with development of delirium in geriatric inpatients: transfer between hospital wards (odds ratio [OR] =2.78; confidence interval [CI] =1.54–5.01; P=0.001), preexisting dementia (OR =2.29; CI =1.44–3.65; P<0.001), previous delirium incidents (OR =2.23; CI =1.47–3.38; P<0.001), previous fall incidents (OR =1.76; CI =1.17–2.64; P=0.006), and use of proton-pump inhibitors (OR =1.67; CI =1.11–2.53; P=0.014). Conclusion Transfer between hospital wards, preexisting dementia, previous delirium incidents, previous fall incidents, and use of proton-pump inhibitors are predictive of development of delirium in the geriatric inpatient setting. PMID:27103793

  18. Dermatologic infections in cancer patients treated with epidermal growth factor receptor inhibitor therapy.

    PubMed

    Eilers, R E; Gandhi, M; Patel, J D; Mulcahy, M F; Agulnik, M; Hensing, T; Lacouture, Mario E

    2010-01-06

    Patients treated with epidermal growth factor receptor inhibitors (EGFRIs) frequently experience dermatologic toxic effects. Whereas the impact of these effects on quality of life and EGFRI dosing has been described, their impact on physical health has not been ascertained. We examined the prevalence of infections that complicate dermatologic toxic effects of EGFRIs. We used retrospective chart review methods to analyze 221 patients who were treated in the Skin and Eye Reactions to Inhibitors of EGFR and Kinases clinic, a referral clinic for dermatologic toxic effects of cancer therapies. We reviewed results of bacterial cultures, histopathologic assessment of biopsy samples, and immunohistochemical staining of skin specimens for viral pathogens that were recorded in the patients' medical records. Associations between patient demographic and treatment characteristics and the development of infections were examined using the Fisher exact test. All statistical tests were two-sided. Eighty-four (38%) of the 221 patients showed evidence of infection at sites of dermatologic toxic effect. Fifty (22.6%) of the 221 patients had cultures positive for Staphylococcus aureus, and 12 (5.4%) of the 221 patients cultured positive for methicillin-resistant S aureus. Less frequent infections included herpes simplex (3.2%), herpes zoster (1.8%), and dermatophytes (10.4%). The seborrheic region was the most prevalent site of infection, and patients with leukopenia had higher risk for infection than patients who did not have leukopenia (P = .005). Demographic factors and associated treatments were not associated with the occurrence of a dermatologic infection (P > or = .05). Patients with dermatologic toxic effects following treatment with EGFRIs have a high prevalence of cutaneous infections. Most notably, bacterial infections developed at sites previously affected by dermatologic toxic effects, with leukopenic patients being at greater risk.

  19. SAR and QSAR study on the bioactivities of human epidermal growth factor receptor-2 (HER2) inhibitors.

    PubMed

    Qu, D; Yan, A; Zhang, J S

    2017-02-01

    In this paper, structure-activity relationship (SAR, classification) and quantitative structure-activity relationship (QSAR) models have been established to predict the bioactivity of human epidermal growth factor receptor-2 (HER2) inhibitors. For the SAR study, we established six SAR (or classification) models to distinguish highly and weakly active HER2 inhibitors. The dataset contained 868 HER2 inhibitors, which was split into a training set including 580 inhibitors and a test set including 288 inhibitors by a Kohonen's self-organizing map (SOM), or a random method. The SAR models were performed using support vector machine (SVM), random forest (RF) and multilayer perceptron (MLP) methods. Among the six models, SVM models obtained superior results compared with other models. The prediction accuracy of the best model (model 1A) was 90.27% and the Matthews correlation coefficient (MCC) was 0.80 on the test set. For the QSAR study, we chose 286 HER2 inhibitors to establish six quantitative prediction models using MLR, SVM and MLP methods. The correlation coefficient (r) of the best model (model 4B) was 0.92 on the test set. The descriptors analysis showed that HAccN, lone pair electronegativity and π electronegativity were closely related to the bioactivity of HER2 inhibitors.

  20. The role of ATF-2 family transcription factors in adipocyte differentiation: antiobesity effects of p38 inhibitors.

    PubMed

    Maekawa, Toshio; Jin, Wanzhu; Ishii, Shunsuke

    2010-02-01

    ATF-2 is a member of the ATF/CREB family of transcription factors and is activated by stress-activated protein kinases, such as p38. To analyze the physiological role of ATF-2 family transcription factors, we have generated mice with mutations in Atf-2 and Cre-bpa, an Atf-2-related gene. The trans-heterozygotes of both mutants were lean and had reduced white adipose tissue (WAT). ATF-2 and CRE-BPa were required for bone morphogenetic protein 2 (BMP-2)-and p38-dependent induction of peroxisome proliferator-activated receptor gamma2 (PPARgamma2), a key transcription factor mediating adipocyte differentiation. Since stored fat supplies have been recognized as a possible target for antiobesity treatments, we tested whether inhibition of the p38-ATF-2 pathway suppresses adipocyte differentiation and leads to reduced WAT by treating mice with a p38 inhibitor for long periods of time. High-fat diet (HFD)-induced obesity was significantly reduced in mice fed the p38 inhibitor. Furthermore, the p38 inhibitor alleviated HFD-induced insulin resistance. In p38 inhibitor-treated mice, macrophage infiltration into WAT was reduced and the tumor necrosis factor alpha (TNF-alpha) levels were lower than control mice. Thus, p38 inhibitors may provide a novel antiobesity treatment.

  1. Synergistic targeting of Sp1, a critical transcription factor for myeloma cell growth and survival, by panobinostat and proteasome inhibitors

    PubMed Central

    Bat-Erdene, Ariunzaya; Miki, Hirokazu; Oda, Asuko; Nakamura, Shingen; Teramachi, Jumpei; Amachi, Ryota; Tenshin, Hirofumi; Hiasa, Masahiro; Iwasa, Masami; Harada, Takeshi; Fujii, Shiro; Sogabe, Kimiko; Kagawa, Kumiko; Yoshida, Sumiko; Endo, Itsuro; Aihara, Kenichi; Abe, Masahiro

    2016-01-01

    Panobinostat, a pan-deacetylase inhibitor, synergistically elicits cytotoxic activity against myeloma (MM) cells in combination with the proteasome inhibitor bortezomib. Because precise mechanisms for panobinostat's anti-MM action still remain elusive, we aimed to clarify the mechanisms of anti-MM effects of panobinostat and its synergism with proteasome inhibitors. Although the transcription factor Sp1 was overexpressed in MM cells, the Sp1 inhibitor terameprocol induced MM cell death in parallel with reduction of IRF4 and cMyc. Panobinostat induced activation of caspase-8, which was inversely correlated with reduction of Sp1 protein levels in MM cells. The panobinostat-mediated effects were further potentiated to effectively induce MM cell death in combination with bortezomib or carfilzomib even at suboptimal concentrations as a single agent. Addition of the caspase-8 inhibitor z-IETD-FMK abolished the Sp1 reduction not only by panobinostat alone but also by its combination with bortezomib, suggesting caspase-8-mediated Sp1 degradation. The synergistic Sp1 reduction markedly suppressed Sp1-driven prosurvival factors, IRF4 and cMyc. Besides, the combinatory treatment reduced HDAC1, another Sp1 target, in MM cells, which may potentiate HDAC inhibition. Collectively, caspase-8-mediated post-translational Sp1 degradation appears to be among major mechanisms for synergistic anti-MM effects of panobinostat and proteasome inhibitors in combination. PMID:27738323

  2. Predictive factor and antihypertensive usage of tyrosine kinase inhibitor-induced hypertension in kidney cancer patients

    PubMed Central

    IZUMI, KOUJI; ITAI, SHINGO; TAKAHASHI, YOSHIKO; MAOLAKE, AERKEN; NAMIKI, MIKIO

    2014-01-01

    Hypertension (HT) is the common adverse event associated with vascular endothelial growth factor receptor-tyrosine kinase inhibitors (VEGFR-TKI). The present study was performed to identify the predictive factors of TKI-induced HT and to determine the classes of antihypertensive agents (AHTA) that demonstrate optimal efficacy against this type of HT. The charts of 50 cases of patients that had received VEGFR-TKI treatment were retrospectively examined. The association between patient background and TKI-induced HT, and the effect of administering AHTA were analyzed. High systolic blood pressure at baseline was identified to be a predictive factor for HT. In addition, there was no difference observed between calcium channel blockers (CCBs) and angiotensin receptor II blockers (ARBs) as first-line AHTA for the control of HT. The findings of the present study may aid with predicting the onset of TKI-induced HT, as well as for its management via the primary use of either CCBs or ARBs. PMID:24959266

  3. Identifying novel factor XIIa inhibitors with PCA-GA-SVM developed vHTS models.

    PubMed

    Chen, Jonathan Jun Feng; Visco, Donald P

    2017-09-01

    There currently is renewed interest in blood clotting Factor XII as a potential target for thrombosis inhibition. Historically untargeted, there is little drug information with which to start drug candidate searches. Typical high-throughput screening can identify potential drug candidates, but is inefficient. Virtual high-throughput screening can be used to raise efficiency by focusing experimental efforts on compounds predicted to be active and is applied here to identify new Factor XIIa inhibitors. We combine principal component analysis, genetic algorithm and support vector machine to create the models used in the virtual high-throughput screening. In this work, experimental data from a PubChem Bioassay was used to train predictive models of Factor XIIa inhibition activity. The models created were then used to virtually screen the entire 72 million PubChem Compound database. Experimental validation of select candidates identified by this process resulted in a 42.9% hit-rate in the first-pass and 100% hit-rate in the second-pass, suggesting the effectiveness of the approach. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

  4. Mechanisms of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Resistance and Strategies to Overcome Resistance in Lung Adenocarcinoma

    PubMed Central

    Chang, Yoon Soo; Choi, Chang-Min

    2016-01-01

    Somatic mutations that lead to hyperactivation of epidermal growth factor receptor (EGFR) signaling are detected in approximately 50% of lung adenocarcinoma in people from the Far East population and tyrosine kinase inhibitors are now the standard first line treatment for advanced disease. They have led to a doubling of progression-free survival and an increase in overall survival by more than 2 years. However, emergence of resistant clones has become the primary cause for treatment failure, and has created a new challenge in the daily management of patients with EGFR mutations. Identification of mechanisms leading to inhibitor resistance has led to new therapeutic modalities, some of which have now been adapted for patients with unsuccessful tyrosine kinase inhibitor treatment. In this review, we describe mechanisms of tyrosine kinase inhibitor resistance and the available strategies to overcoming resistance. PMID:27790276

  5. Platelet-Derived Short-Chain Polyphosphates Enhance the Inactivation of Tissue Factor Pathway Inhibitor by Activated Coagulation Factor XI

    PubMed Central

    Puy, Cristina; Tucker, Erik I.; Ivanov, Ivan S.; Gailani, David; Smith, Stephanie A.; Morrissey, James H.; Gruber, András; McCarty, Owen J. T.

    2016-01-01

    Introduction Factor (F) XI supports both normal human hemostasis and pathological thrombosis. Activated FXI (FXIa) promotes thrombin generation by enzymatic activation of FXI, FIX, FX, and FV, and inactivation of alpha tissue factor pathway inhibitor (TFPIα), in vitro. Some of these reactions are now known to be enhanced by short-chain polyphosphates (SCP) derived from activated platelets. These SCPs act as a cofactor for the activation of FXI and FV by thrombin and FXIa, respectively. Since SCPs have been shown to inhibit the anticoagulant function of TFPIα, we herein investigated whether SCPs could serve as cofactors for the proteolytic inactivation of TFPIα by FXIa, further promoting the efficiency of the extrinsic pathway of coagulation to generate thrombin. Methods and Results Purified soluble SCP was prepared by size-fractionation of sodium polyphosphate. TFPIα proteolysis was analyzed by western blot. TFPIα activity was measured as inhibition of FX activation and activity in coagulation and chromogenic assays. SCPs significantly accelerated the rate of inactivation of TFPIα by FXIa in both purified systems and in recalcified plasma. Moreover, platelet-derived SCP accelerated the rate of inactivation of platelet-derived TFPIα by FXIa. TFPIα activity was not affected by SCP in recalcified FXI-depleted plasma. Conclusions Our data suggest that SCP is a cofactor for TFPIα inactivation by FXIa, thus, expanding the range of hemostatic FXIa substrates that may be affected by the cofactor functions of platelet-derived SCP. PMID:27764259

  6. 76 FR 80829 - Special Conditions: XtremeAir GmbH, XA42; Acrobatic Category Aerodynamic Stability

    Federal Register 2010, 2011, 2012, 2013, 2014

    2011-12-27

    ... airplane. The XA42 airplane has a novel or unusual design feature associated with its static stability... acrobatic capability: Neutral longitudinal and lateral static stability characteristics. Discussion The Code of Federal Regulations states static stability criteria for longitudinal, lateral, and...

  7. The M358R variant of α{sub 1}-proteinase inhibitor inhibits coagulation factor VIIa

    SciTech Connect

    Sheffield, William P.; Bhakta, Varsha

    2016-02-12

    The naturally occurring M358R mutation of the plasma serpin α{sub 1}-proteinase inhibitor (API) changes both its cleavable reactive centre bond to Arg–Ser and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an Arg–Ser reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assays of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparin-independent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 × 10{sup 2} M{sup −1}sec{sup −1}. We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin. - Highlights: • The inhibitory specificity of the serpin alpha-1-proteinase inhibitor (API) is sharply altered in the M358R variant. • API M358R forms denaturation-resistant complexes with coagulation factor VIIa at a rate accelerated by tissue factor but unaffected by heparin. • Complex formation was shown by gel-based assays and quantified kinetically by inhibition of FVIIa-dependent amidolysis.

  8. Fast rotation of a sub-km-sized near-Earth object 2011 XA_3

    NASA Astrophysics Data System (ADS)

    Urakawa, S.; Ohtsuka, K.; Abe, S.; Ito, T.; Nakamura, T.

    2014-07-01

    We present lightcurve observations and multiband photometry for the near-Earth object (NEO) 2011 XA{_3} [1]. Previous studies had shown that most asteroids with rotation periods of < 2.2 h were smaller than 200 m in diameter [2]. The fast-rotating asteroids have structurally significant tensile strength (so-called monolithic asteroids) because such asteroids need to overcome their own centrifugal force. On the other hand, the slow-rotating asteroids larger than 200 m in diameter maintain themselves by their gravity or the cohesive force of bonded aggregates [3], which generally take on a rubble-pile structure [4]. According to a numerical simulation, the fast-rotating asteroids are provided by the collisional disruptions of parent bodies [5]. In-situ observations of the Rosetta spacecraft have revealed that the size frequency distribution of the boulders on (21) Lutetia become shallower in a region of < 150 m in size [6]. When we regard that the size frequency distribution of > 150 m-sized boulders is corresponding to that of the impact ejecta, some ejecta of < 150 m in size may escape from the parent body and likely become small and fast-rotating asteroids. To confirm this hypothesis, we must accumulate more observational data of such asteroids. The purpose of our study is to obtain the rotational period and taxonomic class of the NEO 2011 XA{_3}. The absolute magnitude H = 20.402 ± 0.399 (JPL Small-Body database) of 2011 XA{_3} implies the asteroid to be a subkilometer-sized one. The orbital parameters, a=1.48 au, e=0.93, i=28°, Ω=273°, and ω=323°, are apparently similar to those of the NEO (3200) Phaethon, a=1.27 au, e=0.89, i=22°, Ω=265°, and ω=322°, though indicating a somewhat large difference in the orbital energy. Phaethon is a B/F-type asteroid and probably the main body among the Phaethon-Geminid complex (PGC). The NEOs 2005 UD (F-type) and 1999 YC (C-type) are the other candidates of the PGC along with the Geminid meteor stream [7,8]. We also

  9. Tannic acid, a potent inhibitor of epidermal growth factor receptor tyrosine kinase.

    PubMed

    Yang, Er Bin; Wei, Liu; Zhang, Kai; Chen, Yu Zong; Chen, Wei Ning

    2006-03-01

    Increasing evidence supports the hypothesis that tannic acid, a plant polyphenol, exerts anticarcinogenic activity in chemically induced cancers. In the present study, tannic acid was found to strongly inhibit tyrosine kinase activity of epidermal growth factor receptor (EGFr) in vitro (IC50 = 323 nM). In contrast, the inhibition by tannic acid of p60(c-src) tyrosine kinase (IC50 = 14 microM) and insulin receptor tyrosine kinase (IC50 = 5 microM) was much weaker. The inhibition of EGFr tyrosine kinase by tannic acid was competitive with respect to ATP and non-competitive with respect to peptide substrate. In cultured cells, growth factor-induced tyrosine phosphorylation of growth factor receptors, including EGFr, platelet-derived growth factor receptor, and basic fibroblast growth factor receptor, was inhibited by tannic acid. No inhibition of insulin-induced tyrosine phosphorylation of insulin receptor and insulin-receptor substrate-1 was observed. EGF-stimulated growth of HepG2 cells was inhibited in the presence of tannic acid. The inhibition of serine/threonine-specific protein kinases, including cAMP-dependent protein kinase, protein kinase C and mitogen-activated protein kinase, by tannic acid was only detected at relatively high concentration, IC50 being 3, 325 and 142 microM respectively. The molecular modeling study suggested that tannic acid could be docked into the ATP binding pockets of either EGFr or insulin receptor. These results demonstrate that tannic acid is an in vitro potent inhibitor of EGFr tyrosine kinase.

  10. The First Test Flight of the Delta Clipper-Experimental Advanced (DC-XA)

    NASA Technical Reports Server (NTRS)

    1966-01-01

    The Delta Clipper-Experimental Advanced (DC-XA) is a single-stage-to-orbit, vertical takeoff / vertical landing launch vehicle concept, whose development was geared to significantly reduce launch cost and provided a test bed for NASA Reusable Launch Vehicle (RLV) technology. This photograph shows the descending vehicle landing during the first successful test flight at White Sands Missile Range, New Mexico. The program was discontinued in 2003.

  11. Acoustic Emission Monitoring of the DC-XA Composite Liquid Hydrogen Tank During Structural Testing

    NASA Technical Reports Server (NTRS)

    Wilkerson, C.

    1996-01-01

    The results of acoustic emission (AE) monitoring of the DC-XA composite liquid hydrogen tank are presented in this report. The tank was subjected to pressurization, tensile, and compressive loads at ambient temperatures and also while full of liquid nitrogen. The tank was also pressurized with liquid hydrogen. AE was used to monitor the tank for signs of structural defects developing during the test.

  12. Materials Testing on the DC-X and DC-XA

    NASA Technical Reports Server (NTRS)

    Smith, Dane; Carroll, Carol; Marschall, Jochen; Pallix, Joan

    1997-01-01

    Flight testing of thermal protection materials has been carried out over a two year period on the base heat shield of the Delta Clipper (DC-X and DC-XA), as well on a body flap. The purpose was to use the vehicle as a test bed for materials and more efficient repair or maintenance processes which would be potentially useful for application on new entry vehicles (i.e., X-33, RLV, planetary probes), as well as on the existing space shuttle orbiters. Panels containing Thermal Protection Systems (TPS) and/or structural materials were constructed either at NASA Ames Research Center or at McDonnell Douglas Aerospace (MDA) and attached between two of the four thrusters in the base heat shield of the DC-X or DC-XA. Three different panels were flown on DC-X flights 6, 7, and 8. A total of 7 panels were flown on DC-XA flights 1, 2, and 3. The panels constructed at Ames contained a variety of ceramic TPS including flexible blankets, tiles with high emissivity coatings, lightweight ceramic ablators and other ceramic composites. The MDS test panels consisted primarily of a variety of metallic composites. This report focuses on the ceramic TPS test results.

  13. Comparative nutritional compositions and proteomics analysis of transgenic Xa21 rice seeds compared to conventional rice.

    PubMed

    Gayen, Dipak; Paul, Soumitra; Sarkar, Sailendra Nath; Datta, Swapan K; Datta, Karabi

    2016-07-15

    Transgenic rice expressing the Xa21 gene have enhanced resistant to most devastating bacterial blight diseases caused by Xanthomonas oryzae pv. oryzae (Xoo). However, identification of unintended modifications, owing to the genetic modification, is an important aspect of transgenic crop safety assessment. In this study, the nutritional compositions of seeds from transgenic rice plants expressing the Xa21 gene were compared against non-transgenic rice seeds. In addition, to detect any changes in protein translation levels as a result of Xa21 gene expression, rice seed proteome analyses were also performed by two-dimensional gel electrophoresis. No significant differences were found in the nutritional compositions (proximate components, amino acids, minerals, vitamins and anti-nutrients) of the transgenic and non-transgenic rice seeds. Although gel electrophoresis identified 11 proteins that were differentially expressed between the transgenic and non-transgenic seed, only one of these (with a 20-fold up-regulation in the transgenic seed) shows nutrient reservoir activity. No new toxins or allergens were detected in the transgenic seeds.

  14. Cardenolides from Calotropis gigantea as potent inhibitors of hypoxia-inducible factor-1 transcriptional activity.

    PubMed

    Parhira, Supawadee; Zhu, Guo-Yuan; Chen, Ming; Bai, Li-Ping; Jiang, Zhi-Hong

    2016-12-24

    Calotropis gigantea (L.) Dryand (Apocynaceae) is a medicinal plant native to southern China, India and Southeast Asia. It has been traditionally used for the treatment of several diseases including cancers in these countries. This study aimed to isolate bioactive cardenolides from C. gigantea, to screen their hypoxia-inducible factor (HIF-) 1 inhibitory activity, and to analyze the structure-activity relationship (SAR). Isolation and purification of cardenolides from the latex and the fruits of C. gigantea were performed by using a series of separation techniques. Their structures were fully characterized by elucidating their NMR and HRMS data. The HIF-1 inhibitory activities of cardenolides were evaluated by using a T47D cell-based dual-luciferase reporter assay. The potent cardenolides were selected to further evaluate their dose-response manner. Cytotoxic effects of selected cardenolides were also examined against breast cancer cell line (MCF-7) and normal mammary epithelial cell line (MCF-10A) by MTT assay. Among twenty isolated cardenolides, compounds 1, 3, 4, 6-8, 14 and 17 exhibited stronger HIF-1 inhibitory activities than that of digoxin, a well-known HIF-1 inhibitor (P<0.001). These eight cardenolides inhibited HIF-1 transcriptional activity in a dose-dependent manner with IC50 values in nanomolar potency (21.8-64.9nM). An analysis of SAR revealed the great contributions of a β-configuration of the substituents at positions of C-2' and C-3', an aldehydic moiety on C-19, and the dioxane moiety between the aglycone and sugar parts of cardenolides to the HIF-1 inhibitory activity. In contrast, a hydroxyl group at any positions of C-15, C-16 and C-4' of cardenolides showed negative effects on suppressing HIF-1 transcriptional activity. In addition, these eight cardenolides also exhibited potent cytotoxic effects against human breast cancer cell MCF-7 (IC50 values ranged from 30.5 to 68.8nM), but less toxic effects to human normal mammary epithelial cell MCF

  15. Treatment with didemnin B, an elongation factor 1A inhibitor, improves hepatic lipotoxicity in obese mice.

    PubMed

    Hetherington, Alexandra M; Sawyez, Cynthia G; Sutherland, Brian G; Robson, Debra L; Arya, Rigya; Kelly, Karen; Jacobs, René L; Borradaile, Nica M

    2016-09-01

    Eukaryotic elongation factor EEF1A1 is induced by oxidative and ER stress, and contributes to subsequent cell death in many cell types, including hepatocytes. We recently showed that blocking the protein synthesis activity of EEF1A1 with the peptide inhibitor, didemnin B, decreases saturated fatty acid overload-induced cell death in HepG2 cells. In light of this and other recent work suggesting that limiting protein synthesis may be beneficial in treating ER stress-related disease, we hypothesized that acute intervention with didemnin B would decrease hepatic ER stress and lipotoxicity in obese mice with nonalcoholic fatty liver disease (NAFLD). Hyperphagic male ob/ob mice were fed semipurified diet for 4 weeks, and during week 5 received i.p. injections of didemnin B or vehicle on days 1, 4, and 7. Interestingly, we observed that administration of this compound modestly decreased food intake without evidence of illness or distress, and thus included an additional control group matched for food consumption with didemnin B-treated animals. Treatment with didemnin B improved several characteristics of hepatic lipotoxicity to a greater extent than the effects of caloric restriction alone, including hepatic steatosis, and some hepatic markers of ER stress and inflammation (GRP78, Xbp1s, and Mcp1). Plasma lipid and lipoprotein profiles and histopathological measures of NAFLD, including lobular inflammation, and total NAFLD activity score were also improved by didemnin B. These data indicate that acute intervention with the EEF1A inhibitor, didemnin B, improves hepatic lipotoxicity in obese mice with NAFLD through mechanisms not entirely dependent on decreased food intake, suggesting a potential therapeutic strategy for this ER stress-related disease.

  16. Captopril attenuates hypertension and renal injury induced by the vascular endothelial growth factor inhibitor sorafenib.

    PubMed

    Nagasawa, Tasuku; Hye Khan, Md Abdul; Imig, John D

    2012-05-01

    Vascular endothelial growth factor inhibitors (VEGFi) are known to cause hypertension and renal injury that severely limits their use as an anticancer therapy. We hypothesized that the angiotensin-converting enzyme inhibitor captopril not only prevents hypertension, but also decreases renal injury caused by the VEGFi sorafenib. Rats were administered sorafenib (20 mg/kg per day) alone or in combination with captopril (40 mg/kg per day) for 4 weeks. Sorafenib administration increased blood pressure, which plateaued by day 10. Concurrent treatment with captopril for 4 weeks resulted in a 30 mmHg decrease in blood pressure compared with sorafenib alone (155 ± 5 vs 182 ± 6 mmHg, respectively; P < 0.05). Furthermore, concurrent captopril treatment reduced albuminuria by 50% compared with sorafenib alone (20 ± 8 vs 42 ± 9 mg/day, respectively; P < 0.05) and reduced nephrinuria by eightfold (280 ± 96 vs 2305 ± 665 μg/day, respectively; P < 0.05). Glomerular injury, thrombotic microangiopathy and tubular cast formation were also decreased in captopril-treated rats administered sorafenib. Renal autoregulatory efficiency was determined by evaluating the afferent arteriolar constrictor response to ATP. Sorafenib administration attenuated the vasoconstriction to ATP, whereas concurrent captopril treatment improved ATP reactivity. In conclusion, captopril attenuated hypertension and renal injury and improved renal autoregulatory capacity in rats administered sorafenib. These findings indicate that captopril treatment, in addition to alleviating the detrimental side-effect of hypertension, decreases the renal injury associated with anticancer VEGFi therapies such as sorafenib.

  17. A new class of isothiocyanate-based irreversible inhibitors of macrophage migration inhibitory factor.

    PubMed

    Ouertatani-Sakouhi, Hajer; El-Turk, Farah; Fauvet, Bruno; Roger, Thierry; Le Roy, Didier; Karpinar, Damla Pinar; Leng, Lin; Bucala, Richard; Zweckstetter, Markus; Calandra, Thierry; Lashuel, Hilal A

    2009-10-20

    Macrophage migration inhibitory factor (MIF) is a homotrimeric multifunctional proinflammatory cytokine that has been implicated in the pathogenesis of several inflammatory and autoimmune diseases. Current therapeutic strategies for targeting MIF focus on developing inhibitors of its tautomerase activity or modulating its biological activities using anti-MIF neutralizing antibodies. Herein we report a new class of isothiocyanate (ITC)-based irreversible inhibitors of MIF. Modification by benzyl isothiocyanate (BITC) and related analogues occurred at the N-terminal catalytic proline residue without any effect on the oligomerization state of MIF. Different alkyl and arylalkyl ITCs modified MIF with nearly the same efficiency as BITC. To elucidate the mechanism of action, we performed detailed biochemical, biophysical, and structural studies to determine the effect of BITC and its analogues on the conformational state, quaternary structure, catalytic activity, receptor binding, and biological activity of MIF. Light scattering, analytical ultracentrifugation, and NMR studies on unmodified and ITC-modified MIF demonstrated that modification of Pro1 alters the tertiary, but not the secondary or quaternary, structure of the trimer without affecting its thermodynamic stability. BITC induced drastic effects on the tertiary structure of MIF, in particular residues that cluster around Pro1 and constitute the tautomerase active site. These changes in tertiary structure and the loss of catalytic activity translated into a reduction in MIF receptor binding activity, MIF-mediated glucocorticoid overriding, and MIF-induced Akt phosphorylation. Together, these findings highlight the role of tertiary structure in modulating the biochemical and biological activities of MIF and present new opportunities for modulating MIF biological activities in vivo.

  18. Selective serotonin reuptake inhibitor discontinuation: side effects and other factors that influence medication adherence.

    PubMed

    Goethe, John W; Woolley, Stephen B; Cardoni, Alex A; Woznicki, Brenda A; Piez, Deborah A

    2007-10-01

    Patients with major depression discontinue taking their antidepressants for many reasons. Although side effects are often cited as the reason for discontinuation, few prospective studies have addressed this question, and none has specifically examined discontinuation in patients with severe depression. Inpatients and outpatients treated with a selective serotonin reuptake inhibitor for major depressive disorder were identified after admission. Three months later, patients were contacted and interviewed to determine antidepressant usage and the side effects experienced, including when these were experienced and their severity. Between October 2001 and April 2003, 406 English- or Spanish-speaking patients aged 18 to 75 years were followed up. One in 4 patients discontinued the index antidepressant. Among specific side effects noted, only "change in weight" and "anxiety" were significant predictors of discontinuation after controlling for confounders. Experiencing 1 or more "extremely" bothersome side effects was associated with more than a doubling of the risk of discontinuation, but the presence of side effects and side effects less severe than "extremely" bothersome were not significant predictors. There were no differences among selective serotonin reuptake inhibitor antidepressants in either the presence/absence of side effects or in the discontinuation rates. The results suggest that the contribution of side effects to antidepressant discontinuation is more complex than previously suggested. Disparate findings from earlier studies may reflect aspects of study design, such as examining populations whose severity of depression varied widely or not controlling for important confounding factors. Future research should separately examine high-risk groups (or control for severity of depression) and carefully rule out other potential contributors to discontinuation.

  19. Captopril attenuates hypertension and renal injury induced by the vascular endothelial growth factor inhibitor sorafenib

    PubMed Central

    Nagasawa, Tasuku; Khan, Abdul Hye; Imig, John D

    2013-01-01

    SUMMARY Vascular endothelial growth factor inhibitors (VEGFi) are known to cause hypertension and renal injury that severely limits their use as an anticancer therapy. We hypothesized that the angiotensin-converting enzyme inhibitor captopril not only prevents hypertension, but also decreases renal injury caused by the VEGFi sorafenib.Rats were administered sorafenib (20 mg/kg per day) alone or in combination with captopril (40 mg/kg per day) for 4 weeks. Sorafenib administration increased blood pressure, which plateaued by day 10.Concurrent treatment with captopril for 4 weeks resulted in a 30 mmHg decrease in blood pressure compared with sorafenib alone (155 ± 5 vs 182 ± 6 mmHg, respectively; P < 0.05). Furthermore, concurrent captopril treatment reduced albuminuria by 50% compared with sorafenib alone (20 ± 8 vs 42 ± 9 mg/day, respectively; P < 0.05) and reduced nephrinuria by eightfold (280 ± 96 vs 2305 ± 665 μg/day, respectively; P < 0.05). Glomerular injury, thrombotic micro-angiopathy and tubular cast formation were also decreased in captopril-treated rats administered sorafenib. Renal autoregulatory efficiency was determined by evaluating the afferent arteriolar constrictor response to ATP. Sorafenib administration attenuated the vasoconstriction to ATP, whereas concurrent captopril treatment improved ATP reactivity.In conclusion, captopril attenuated hypertension and renal injury and improved renal autoregulatory capacity in rats administered sorafenib. These findings indicate that captopril treatment, in addition to alleviating the detrimental side-effect of hypertension, decreases the renal injury associated with anticancer VEGFi therapies such as sorafenib. PMID:22443474

  20. Kirromycin, an Inhibitor of Protein Biosynthesis that Acts on Elongation Factor Tu

    PubMed Central

    Wolf, Heinz; Chinali, Gianni; Parmeggiani, Andrea

    1974-01-01

    Kirromycin, a new inhibitor of protein synthesis, is shown to interfere with the peptide transfer reaction by acting on elongation factor Tu (EF-Tu). All the reactions associated with this elongation factor are affected. Formation of the EF-Tu·GTP complex is strongly stimulated. Peptide bond formation is prevented only when Phe-tRNAPhe is bound enzymatically to ribosomes, presumably because GTP hydrolysis associated with enzymatic binding of Phe-tRNAPhe is not followed by release of EF-Tu·GDP from the ribosome. This antibiotic also enables EF-Tu to catalyze the binding of Phe-tRNAPhe to the poly(U)·ribosome complex even in the absence of GTP. EF-Tu activity in the GTPase reaction is dramatically affected by kirromycin: GTP hydrolysis, which normally requires ribosomes and aminoacyl-tRNA, takes place with the elongation factor alone. This GTPase shows the same Km for GTP as the one dependent on Phe-tRNAPhe and ribosomes in the absence of the antibiotic. Ribosomes and Phe-tRNAPhe, but not tRNAPhe or Ac-Phe-tRNAPhe, stimulate the kirromycin-induced EF-Tu GTPase. These results indicate that the catalytic center of EF-Tu GTPase that is dependent upon aminoacyl-tRNA and ribosomes is primarily located on the elongation factor. In conclusion, kirromycin can substitute for GTP, aminoacyl-tRNA, or ribosomes in various reactions involving EF-Tu, apparently by affecting the allosteric controls between the sites on the EF-Tu molecule interacting with these components. PMID:4373734

  1. Effect of Tumor Necrosis Factor Inhibitor Therapy on Osteoclasts Precursors in Rheumatoid Arthritis

    PubMed Central

    Rodrigues, Ana Maria; Campanilho-Marques, Raquel; Ponte, Cristina; Canhão, Helena; Ainola, Mari

    2017-01-01

    Objective. Tumor necrosis factor (TNF) increases circulating osteoclast (OC) precursors numbers by promoting their proliferation and differentiation. The aim of this study was to assess the effect of TNF inhibitors (TNFi) on the differentiation and activity of OC in rheumatoid arthritis (RA) patients. Methods. Seventeen RA patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers, in vitro OC differentiation assays, and qRT-PCR for OC specific genes was performed. Results. After TNFi therapy, patients had reduced RANKL surface expression in B-lymphocytes and the frequency of circulating classical CD14brightCD16− monocytes was decreased. Serum levels of sRANKL, sRANKL/OPG ratio, and CTX-I were reduced in RA patients after TNFi treatment. Moreover, after exposure to TNFi, osteoclast differentiation and activity were decreased, as well as the expression of TRAF6 and cathepsin K. Conclusion. We propose that TNFi arrests bone loss and erosion, through two pathways: direct reduction of osteoclast precursor numbers and inhibition of intracellular signaling pathways acting through TRAF6. PMID:28286757

  2. Antiangiogenic mechanisms of PJ-8, a novel inhibitor of vascular endothelial growth factor receptor signaling.

    PubMed

    Huang, Shiu-Wen; Lien, Jin-Cherng; Kuo, Sheng-Chu; Huang, Tur-Fu

    2012-05-01

    Angiogenesis occurs not only during tissue growth and development but also during wound healing and tumor progression. Angiogenesis is a balanced process controlled by proangiogenic and antiangiogenic molecules. As a critical factor in the induction of angiogenesis, vascular endothelial growth factor (VEGF) has become an attractive target for antiangiogenic and cancer therapeutic agents. In an effort to develop novel inhibitors to block VEGF signaling, we selected Pj-8, a benzimidazole derivative, and investigated its inhibitory mechanisms in human umbilical vascular endothelial cells (HUVECs). Pj-8 concentration-dependently inhibited VEGF-induced proliferation, migration and tube formation of HUVECs. Pj-8 also suppressed VEGF-induced microvessel sprouting from aortic rings ex vivo and suppressed neovascularization of implanted matrigel plugs in vivo. Pj-8 inhibited VEGF-induced phosphorylation of VEGF receptor (VEGFR) 2 and the downstream protein kinases, including Akt, focal adhesion kinase, extracellular signal-regulated kinases and Src. Results from in vitro kinase assay further demonstrated that Pj-8 suppressed the kinase activity of 3-phosphoinositide-dependent kinase 1 (PDK1). Using xenograft tumor angiogenesis model, Pj-8 markedly eliminated tumor-associated angiogenesis. Taken together, our findings suggest that Pj-8 inhibits VEGF and tumor cells MDA-MB-231-induced angiogenesis, and it may be a potential drug candidate in anticancer therapy. Downregulation of VEGFR2-mediated signaling may contribute to its antiangiogenic actions.

  3. Design and Synthesis of Novel Small-molecule Inhibitors of the Hypoxia Inducible Factor Pathway

    PubMed Central

    Mooring, Suazette Reid; Jin, Hui; Devi, Narra S.; Jabbar, Adnan A.; Kaluz, Stefan; Liu, Yuan; Van Meir, Erwin G.; Wang, Binghe

    2012-01-01

    Hypoxia, a reduction in partial oxygen pressure, is a salient property of solid tumors. Hypoxia drives malignant progression and metastasis in tumors and participates in tumor resistance to radio- and chemotherapies. Hypoxia activates the hypoxia-inducible factor (HIF) family of transcription factors, which induce target genes that regulate adaptive biological processes such as anaerobic metabolism, cell motility and angiogenesis. Clinical evidence has demonstrated that expression of HIF-1 is strongly associated with poor patient prognosis and activation of HIF-1 contributes to malignant behavior and therapeutic resistance. Consequently, HIF-1 has become an important therapeutic target for inhibition by small molecules. Herein, we describe the design and synthesis of small molecules that inhibit the HIF-1 signaling pathway. Many of these compounds exhibit inhibitory activity in the nanomolar range. Separate mechanistic studies indicate that these inhibitors do not alter HIF-1 levels, but interfere with the HIF-1α/HIF-1β/p300/CBP complex formation by interacting with p300 and CBP. PMID:22032632

  4. A nuclear factor-κB inhibitor, dehydroxymethylepoxyquinomicin, ameliorates GVHD in allogeneic bone marrow transplantation.

    PubMed

    Yamanouchi, Sohsaku; Adachi, Yasushi; Shimo, Tomohiko; Umezawa, Kazuo; Okigaki, Mitsuhiko; Tsuji, Shoji; Li, Ming; Takaya, Junji; Kuge, Tomohiro; Ikehara, Susumu; Kaneko, Kazunari

    2015-09-01

    GVHD is a crucial mortality factor in allogeneic bone marrow transplantation (ABMT). In this paper, we show that dehydroxymethylepoxyquinomicin (DHMEQ), a novel inhibitor of nuclear factor-κB, suppresses GVHD, resulting in an improved mortality rate in a mouse ABMT model. Bone marrow cells from C57BL/6 mice (B6 mice) were transplanted into lethally irradiated BALB/c mice. Two weeks later, spleen cells from B6 mice were transplanted into the irradiated BALB/c mice. From one week after the injection of spleen cells, when the mice started to show GVHD, the mice were also injected intraperitoneally daily with DHMEQ or vehicle only (DMSO) for 4 weeks. By 80 days after the ABMT, 6/14 of the vehicle-injected mice (43%) had died because of GVHD, whereas all DHMEQ-injected mice survived this observation period and developed milder GVHD than the vehicle-injected mice. When regulatory T cells were reduced by the injection of anti-folate receptor 4 (FR4) antibody, the effects of DHMEQ were reduced. These findings suggest that administration of DHMEQ could become a new strategy for preventing fatalities from GVHD. Copyright © 2015 Elsevier GmbH. All rights reserved.

  5. Epidermal growth factor receptor inhibitors trigger a type I interferon response in human skin

    PubMed Central

    Pastore, Saveria

    2016-01-01

    The Epidermal Growth Factor Receptor (EGFR) is centrally involved in the regulation of key processes of the epithelia, including cell proliferation, survival, differentiation, and also tumorigenesis. Humanized antibodies and small-molecule inhibitors targeting EGFR were developed to disrupt these functions in cancer cells and are currently used in the treatment of diverse metastatic epithelial cancers. By contrast, these drugs possess significant skin-specific toxic effects, comprising the establishment of a persistent inflammatory milieu. So far, the molecular mechanisms underlying these epiphenomena have been investigated rather poorly. Here we showed that keratinocytes respond to anti-EGFR drugs with the development of a type I interferon molecular signature. Upregulation of the transcription factor IRF1 is early implicated in the enhanced expression of interferon-kappa, leading to persistent activation of STAT1 and further amplification of downstream interferon-induced genes, including anti-viral effectors and chemokines. When anti-EGFR drugs are associated to TNF-α, whose expression is enhanced by the drugs themselves, all these molecular events undergo a dramatic enhancement by synergy mechanisms. Finally, high levels of interferon-kappa can be observed in epidermal keratinocytes and also in leukocytes infiltrating the upper dermis of cetuximab-driven skin lesions. Our data suggest that dysregulated activation of type I interferon innate immunity is implicated in the molecular processes triggered by anti-EGFR drugs and leading to persistent skin inflammation. PMID:27322144

  6. Endogenous tissue factor pathway inhibitor has a limited effect on host defence in murine pneumococcal pneumonia.

    PubMed

    van den Boogaard, Florry E; van 't Veer, Cornelis; Roelofs, Joris J T H; Meijers, Joost C M; Schultz, Marcus J; Broze, George J; van der Poll, Tom

    2015-07-01

    Streptococcus (S.) pneumoniae is the most common causative pathogen in community-acquired pneumonia. Coagulation and inflammation interact in the host response to infection. Tissue factor pathway inhibitor (TFPI) is a natural anticoagulant protein that inhibits tissue factor (TF), the main activator of inflammation-induced coagulation. It was the objective of this study to investigate the effect of endogenous TFPI levels on coagulation, inflammation and bacterial growth during S. pneumoniae pneumonia in mice. The effect of low endogenous TFPI levels was studied by administration of a neutralising anti-TFPI antibody to wild-type mice, and by using genetically modified mice expressing low levels of TFPI, due to a genetic deletion of the first Kunitz domain of TFPI (TFPIK1(-/-)) rescued with a human TFPI transgene. Pneumonia was induced by intranasal inoculation with S. pneumoniae and samples were obtained at 6, 24 and 48 hours after infection. Anti-TFPI reduced TFPI activity by ~50 %. Homozygous lowTFPI mice and heterozygous controls had ~10 % and ~50 % of normal TFPI activity, respectively. TFPI levels did not influence bacterial growth or dissemination. Whereas lung pathology was unaffected in all groups, mice with ~10 % (but not with ~50 %) of TFPI levels displayed elevated lung cytokine and chemokine concentrations 24 hours after infection. None of the groups with low TFPI levels showed an altered procoagulant response in lungs or plasma during pneumonia. These data argue against an important role for endogenous TFPI in the antibacterial, inflammatory and procoagulant response during pneumococcal pneumonia.

  7. Microbial hydroxylation and glycosylation of pentacyclic triterpenes as inhibitors on tissue factor procoagulant activity.

    PubMed

    Wang, Wei-Wei; Xu, Shao-Hua; Zhao, Ya-Zheng; Zhang, Chao; Zhang, Yuan-Yuan; Yu, Bo-Yang; Zhang, Jian

    2017-02-15

    To discover new inhibitors on tissue factor procoagulant activity, 20 pentacyclic triterpenes were semi-synthetized through microbial transformation and assayed on the model of human THP-1 cells stimulated by lipopolysaccharide. In the biotransformation two types of reactions were observed, regio-selective hydroxylation and glycosylation. The bioassay results showed that most of tested compounds were significant effective on this model and two of the biotransformation products 23-hydroxy-28-O-β-d-glucopyranosyl betulinic acid (3d) and 28-O-β-d-glucopyranosyl oleanic acid (1a) exhibited most potential activities with the IC50 values of 0.028, 0.035nM respectively. The preliminary structure and activity relationship analysis revealed that the aglycones with single free hydroxyl group on the skeleton (1, 1j) were less effective than that with more free hydroxyl groups (1d, 1f, 2), mono-glycosylation can significantly enhance their inhibitory effects. Our findings also provide some potential leading compounds for tissue factor-related diseases, such as cancer and cardiovascular diseases. Copyright © 2017 Elsevier Ltd. All rights reserved.

  8. Prophylaxis and treatment of dermatologic adverse events from epidermal growth factor receptor inhibitors.

    PubMed

    Wu, Peggy A; Balagula, Yevgeniy; Lacouture, Mario E; Anadkat, Milan J

    2011-07-01

    As the number and uses for targeted therapies such as epidermal growth factor receptor inhibitors (EGFRIs) increase, so does the need to recognize and treat the dermatologic side-effects of these agents. Although agents such as gefitinib, erlotinib, cetuximab, lapatinib, and panitumumab have less systemic side-effects than traditional cytotoxic chemotherapy, dermatologic adverse events from EGFRIs are significantly more common. These dermatologic toxicities have previously led to reduction or cessation of therapy and recently have been shown to decrease patients' quality of life. This review provides a symptom-based treatment approach to the common dermatologic adverse effects seen with the epidermal growth factor receptor antagonists: papulopustular rash, xerosis, pruritus as well as hair, nail, and mucosal changes. Each dermatologic toxicity is described; prophylaxis and treatment options, from topical to systemic, are presented based on a review of the current literature with emphasis on new clinical trials results. We also provide specific recommendations based on our practice in a specialty clinic. Although the field continues to evolve, this review presents the most up-to-date information on managing dermatologic adverse effects of EGFRIs. Practitioners should find this article to be a practical resource in approaching patients on EGFRIs with dermatologic toxicities. As we learn how to optimally manage the adverse effects of these agents, we practitioners have the opportunity to increase patients' quality of life and decrease reductions or cessations of life-prolonging therapy.

  9. The potential roles of hepatocyte growth factor (HGF)-MET pathway inhibitors in cancer treatment.

    PubMed

    Parikh, Rahul A; Wang, Peng; Beumer, Jan H; Chu, Edward; Appleman, Leonard J

    2014-01-01

    MET is located on chromosome 7q31 and is a proto-oncogene that encodes for hepatocyte growth factor (HGF) receptor, a member of the receptor tyrosine kinase (RTK) family. HGF, also known as scatter factor (SF), is the only known ligand for MET. MET is a master regulator of cell growth and division (mitogenesis), mobility (motogenesis), and differentiation (morphogenesis); it plays an important role in normal development and tissue regeneration. The HGF-MET axis is frequently dysregulated in cancer by MET gene amplification, translocation, and mutation, or by MET or HGF protein overexpression. MET dysregulation is associated with an increased propensity for metastatic disease and poor overall prognosis across multiple tumor types. Targeting the dysregulated HGF-MET pathway is an area of active research; a number of monoclonal antibodies to HGF and MET, as well as small molecule inhibitors of MET, are under development. This review summarizes the key biological features of the HGF-MET axis, its dysregulation in cancer, and the therapeutic agents targeting the HGF-MET axis, which are in development.

  10. Comparison of Clot-based, Chromogenic, and Fluorescence Assays for Measurement of Factor VIII Inhibitors in the U.S. Hemophilia Inhibitor Research Study

    PubMed Central

    Miller, Connie H.; Rice, Anne S.; Boylan, Brian; Shapiro, Amy D.; Lentz, Steven R.; Wicklund, Brian M.; Kelly, Fiona M.; Soucie, J. Michael

    2015-01-01

    Summary Background Detection and validation of inhibitors (antibodies) to hemophilia treatment products are important for clinical care, evaluation of product safety, and assessment of population trends. Methods Centralized monitoring for factor VIII (FVIII) inhibitors was conducted for patients in the Hemophilia Inhibitor Research Study using a previously reported modified Nijmegen-Bethesda clotting assay (NBA), a chromogenic Bethesda assay (CBA), and a novel fluorescence immunoassay (FLI). Results NBA and CBA were performed on 1005 specimens and FLI on 272 specimens. CBA was negative on 880/883 specimens (99.7%) with Nijmegen-Bethesda units (NBU)<0.5 and positive on 42/42 specimens (100%) with NBU≥2.0 and 43/80 specimens (53.8%) with NBU 0.5–1.9. Among specimens with positive NBA and negative CBA, 58.1% were FLI-negative, 12.9% had evidence of lupus anticoagulant, and 35.5% had non-time-dependent inhibition. CBA and FLI were positive on 72.4% and 100% of 1.0–1.9 NBU specimens and 43.1% and 50.0% of 0.5–0.9 NBU specimens. FLI detected antibodies in 98.0% of CBA-positive and 81.6% of NBA-positive specimens (P=0.004). Among 21 new inhibitors detected by NBA, 5 (23.8%) with 0.7–1.3 NBU did not react in CBA or FLI. Among previously positive patients with 0.5–1.9 NBU, 7/25 (28%) were not CBA or FLI positive. FLI was positive on 36/169 NBU-negative specimens (21.3%). Conclusions FVIII specificity could not be demonstrated by CBA or FLI for 26% of inhibitors of 0.5–1.9 NBU; such results must be interpreted with caution. Low titer inhibitors detected in clot-based assays should always be repeated, with consideration given to evaluating their reactivity with FVIII using more specific assays. PMID:23601690

  11. Intensity of factor VIII treatment and inhibitor development in children with severe hemophilia A: the RODIN study.

    PubMed

    Gouw, Samantha C; van den Berg, H Marijke; Fischer, Kathelijn; Auerswald, Günter; Carcao, Manuel; Chalmers, Elizabeth; Chambost, Hervé; Kurnik, Karin; Liesner, Ri; Petrini, Pia; Platokouki, Helen; Altisent, Carmen; Oldenburg, Johannes; Nolan, Beatrice; Garrido, Rosario Pérez; Mancuso, M Elisa; Rafowicz, Anne; Williams, Mike; Clausen, Niels; Middelburg, Rutger A; Ljung, Rolf; van der Bom, Johanna G

    2013-05-16

    The objective of this study was to examine the association of the intensity of treatment, ranging from high-dose intensive factor VIII (FVIII) treatment to prophylactic treatment, with the inhibitor incidence among previously untreated patients with severe hemophilia A. This cohort study aimed to include consecutive patients with a FVIII activity < 0.01 IU/mL, born between 2000 and 2010, and observed during their first 75 FVIII exposure days. Intensive FVIII treatment of hemorrhages or surgery at the start of treatment was associated with an increased inhibitor risk (adjusted hazard ratio [aHR], 2.0; 95% confidence interval [CI], 1.3-3.0). High-dose FVIII treatment was associated with a higher inhibitor risk than low-dose FVIII treatment (aHR, 2.3; 95% CI, 1.0-4.8). Prophylaxis was only associated with a decreased overall inhibitor incidence after 20 exposure days of FVIII. The association with prophylaxis was more pronounced in patients with low-risk F8 genotypes than in patients with high-risk F8 genotypes (aHR, 0.61, 95% CI, 0.19-2.0 and aHR, 0.85, 95% CI, 0.51-1.4, respectively). In conclusion, our findings suggest that in previously untreated patients with severe hemophilia A, high-dosed intensive FVIII treatment increases inhibitor risk and prophylactic FVIII treatment decreases inhibitor risk, especially in patients with low-risk F8 mutations.

  12. A Fluorescence Polarization Assay for Binding to Macrophage Migration Inhibitory Factor and Crystal Structures for Complexes of Two Potent Inhibitors

    PubMed Central

    2016-01-01

    Human macrophage migration inhibitory factor (MIF) is both a keto–enol tautomerase and a cytokine associated with numerous inflammatory diseases and cancer. Consistent with observed correlations between inhibition of the enzymatic and biological activities, discovery of MIF inhibitors has focused on monitoring the tautomerase activity using l-dopachrome methyl ester or 4-hydroxyphenyl pyruvic acid as substrates. The accuracy of these assays is compromised by several issues including substrate instability, spectral interference, and short linear periods for product formation. In this work, we report the syntheses of fluorescently labeled MIF inhibitors and their use in the first fluorescence polarization-based assay to measure the direct binding of inhibitors to the active site. The assay allows the accurate and efficient identification of competitive, noncompetitive, and covalent inhibitors of MIF in a manner that can be scaled for high-throughput screening. The results for 22 compounds show that the most potent MIF inhibitors bind with Kd values of ca. 50 nM; two are from our laboratory, and the other is a compound from the patent literature. X-ray crystal structures for two of the most potent compounds bound to MIF are also reported here. Striking combinations of protein–ligand hydrogen bonding, aryl–aryl, and cation−π interactions are responsible for the high affinities. A new chemical series was then designed using this knowledge to yield two more strong MIF inhibitors/binders. PMID:27299179

  13. Hemofiltration during cardiopulmonary bypass: the effect on anti-Xa and anti-IIa heparin activity.

    PubMed

    Despotis, G J; Levine, V; Filos, K S; Joiner-Maier, D; Joist, J H

    1997-03-01

    Previous studies have demonstrated that heparin concentrations during cardiopulmonary bypass (CPB) may vary considerably, which may be related to variability in redistribution, cellular and plasma protein binding, and clearance of heparin. The purpose of this study was to determine whether hemofiltration removes lower molecular weight fractions of heparin from plasma and thus contributes to variability of blood levels of heparin. Twenty patients undergoing cardiac surgery with CPB were enrolled in this study after informed consent was obtained. The study was subdivided into two phases. The first 10 patients were enrolled in Phase I, which was designed to determine whether hemofiltration removes lower molecular weight fractions of heparin from blood. Blood specimens obtained from the inflow line and outflow lines of the hemofiltration unit were used to measure complete blood counts (CBC) and plasma heparin activity by anti-Xa and anti-IIa assays. Phase II was designed to evaluate the effect of hemofiltration on circulating plasma heparin activity. In Phase II, blood specimens were obtained from 10 patients via the arterial cannula of the extracorporeal circuit prior to and after hemofiltration for measurements of CBCs, anti-Xa plasma heparin, as well as whole blood heparin concentration using an automated protamine titration assay (Hepcon instrument, Medtronic Inc., Parker, CO). Ultrafiltrate and reservoir volumes were measured in both phases of the study. Hemofiltration did not remove lower (anti-Xa measurable) molecular weight heparin, but it resulted in a considerable increase in heparin activity in the outflow line, as measured by both anti-Xa and anti-IIa assays. The plasma anti-Xa heparin activity obtained after hemofiltration (5 +/- 1.8 U/mL) was substantially (P = 0.003) greater than heparin activity obtained prior to hemofiltration (3.9 +/- 1.7 U/mL). The increase in heparin activity with hemofiltration was directly related to ultrafiltrate volume (r = 0

  14. Concurrent Autophagy Inhibition Overcomes the Resistance of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Human Bladder Cancer Cells

    PubMed Central

    Kang, Minyong; Lee, Kyoung-Hwa; Lee, Hye Sun; Jeong, Chang Wook; Kwak, Cheol; Kim, Hyeon Hoe; Ku, Ja Hyeon

    2017-01-01

    Despite the potential therapeutic efficacy of epithelial growth factor receptor (EGFR) inhibitors in the treatment of advanced stage bladder cancer, there currently is no clear evidence to support this hypothesis. In this study, we investigate whether the concurrent treatment of autophagy-blocking agents with EGFR inhibitors exerts synergistic anti-cancer effects in T24 and J82 human bladder cancer cells. Lapatinib and gefitinib were used as EGFR inhibitors, and bafilomycin A1 (BFA1), chloroquine (CQ) and 3-methyladenine (3-MA) were used as the pharmacologic inhibitors of autophagy activities. To assess the proliferative and self-renewal capabilities, the Cell Counting Kit-8 (CCK-8) assay and a clonogenic assay were performed, respectively. To examine apoptotic cell death, flow cytometry using annexin-V/propidium iodide (PI) was used. To measure the autophagy activities, the expression levels of LC3I and II was determined by Western blot analysis. To validate the synergistic effects of autophagy inhibition with EGFR inhibitors, we specifically blocked key autophagy regulatory gene ATG12 by transfection of small interference RNA and examined the phenotypic changes. Of note, lapatinib and gefitinib triggered autophagy activities in T24 and J82 human bladder cancer cells, as indicated by upregulation of LC3II. More importantly, inhibiting autophagy activities with pharmacologic inhibitors (BFA1, CQ or 3-MA) remarkably reduced the cell viabilities and clonal proliferation of T24 and J82 cells, compared to those treated with either of the agents alone. We also obtained similar results of the enhanced anti-cancer effects of EGFR inhibitors by suppressing the expression of ATG12. Notably, the apoptotic assay showed that synergistic anti-cancer effects were induced via the increase of apoptotic cell death. In summary, concomitant inhibition of autophagy activities potentiated the anti-cancer effects of EGFR inhibitors in human bladder cancer cells, indicating a novel

  15. Joint bleeds increase the inhibitor response to human factor VIII in a rat model of severe haemophilia A.

    PubMed

    Lövgren, K M; Søndergaard, H; Skov, S; Wiinberg, B

    2016-09-01

    The most serious complication in haemophilia A (HA) replacement therapy with coagulation factor VIII (FVIII) is neutralizing antibodies, i.e. inhibitors. It has been hypothesized that danger signals generated during a bleed might have an adjuvant effect on the immune response to FVIII in on-demand treatment, increasing the inhibitor risk. To compare the antibody response to treatment with recombinant human FVIII (rhFVIII) in relation to induced knee joint bleeds and treatment without concurrent bleeds in a HA rat model. HA rats were divided into two groups: one group (n = 10) receiving three needle induced knee joint bleeds 14 days apart and a control group (n = 9) receiving three sham procedures. Three hours after each injury/sham 50 IU kg(-1) rhFVIII was administrated intravenously. Subsequently, both groups continued rhFVIII treatment for another 9 weeks. Binding antibodies were analysed using an enzyme-linked immunosorbent assay and neutralizing antibodies using a Bethesda-like assay. Rats in the knee-bleed group developed a significantly faster inhibitor response and reached significantly higher inhibitor levels. In the knee-bleed group, 80% developed inhibitors vs. 33% in the control group, demonstrating a 2.4 times higher inhibitor risk when treating concurrent with bleeds. FVIII treatment in relation to a bleed potentiates inhibitor development compared to FVIII treatment alone in this HA rat, indicating that bleeding is a potential danger signal. Our results support the theory that FVIII replacement therapy concurrent with a bleeding episode increases the inhibitor risk, which to the best of our knowledge, has not been confirmed in an animal model before. © 2016 John Wiley & Sons Ltd.

  16. Platelets Contain Tissue Factor Pathway Inhibitor-2 Derived from Megakaryocytes and Inhibits Fibrinolysis*

    PubMed Central

    Vadivel, Kanagasabai; Ponnuraj, Sathya-Moorthy; Kumar, Yogesh; Zaiss, Anne K.; Bunce, Matthew W.; Camire, Rodney M.; Wu, Ling; Evseenko, Denis; Herschman, Harvey R.; Bajaj, Madhu S.; Bajaj, S. Paul

    2014-01-01

    Tissue factor pathway inhibitor-2 (TFPI-2) is a homologue of TFPI-1 and contains three Kunitz-type domains and a basic C terminus region. The N-terminal domain of TFPI-2 is the only inhibitory domain, and it inhibits plasma kallikrein, factor XIa, and plasmin. However, plasma TFPI-2 levels are negligible (≤20 pm) in the context of influencing clotting or fibrinolysis. Here, we report that platelets contain significant amounts of TFPI-2 derived from megakaryocytes. We employed RT-PCR, Western blotting, immunohistochemistry, and confocal microscopy to determine that platelets, MEG-01 megakaryoblastic cells, and bone marrow megakaryocytes contain TFPI-2. ELISA data reveal that TFPI-2 binds factor V (FV) and partially B-domain-deleted FV (FV-1033) with Kd ∼9 nm and binds FVa with Kd ∼100 nm. Steady state analysis of surface plasmon resonance data reveal that TFPI-2 and TFPI-1 bind FV-1033 with Kd ∼36–48 nm and bind FVa with Kd ∼252–456 nm. Further, TFPI-1 (but not TFPI-1161) competes with TFPI-2 in binding to FV. These data indicate that the C-terminal basic region of TFPI-2 is similar to that of TFPI-1 and plays a role in binding to the FV B-domain acidic region. Using pull-down assays and Western blots, we show that TFPI-2 is associated with platelet FV/FVa. TFPI-2 (∼7 nm) in plasma of women at the onset of labor is also, in part, associated with FV. Importantly, TFPI-2 in platelets and in plasma of pregnant women inhibits FXIa and tissue-type plasminogen activator-induced clot fibrinolysis. In conclusion, TFPI-2 in platelets from normal or pregnant subjects and in plasma from pregnant women binds FV/Va and regulates intrinsic coagulation and fibrinolysis. PMID:25262870

  17. Induction of an inhibitor antibody to factor XI in a patient with severe inherited factor XI deficiency by Rh immune globulin.

    PubMed

    Zucker, Michal; Zivelin, Ariella; Teitel, Jerome; Seligsohn, Uri

    2008-02-01

    In this paper, we report an inhibitor antibody to factor XI (FXI) in a woman with severe inherited FXI deficiency, induced by FXI present in an Rh immune globulin preparation. The patient is homozygous for the Glu117Stop mutation, associated with a FXI level of less than 1 U/dL. Unlike all previously described patients with severe FXI deficiency and an inhibitor, the patient had never been exposed to blood products. Following 3 injections of Rh immune globulin during pregnancy, she developed an inhibitor to FXI (8 Bethesda units) that was shown to bind specifically to FXI and inhibit factor IX cleavage by purified FXIa. The administered Rh immune globulin and 2 other similar products were shown to contain FXI. Clinicians should be aware of the potential for immunization of severely FXI-deficient patients by FXI present in Rh immune globulin preparations.

  18. Target-mediated clearance and bio-distribution of a monoclonal antibody against the Kunitz-type protease inhibitor 2 domain of Tissue Factor Pathway Inhibitor.

    PubMed

    Hansen, Lene; Petersen, Lars Christian; Lauritzen, Brian; Clausen, Jes Thorn; Grell, Susanne Nedergaard; Agersø, Henrik; Sørensen, Brit Binow; Hilden, Ida; Almholt, Kasper

    2014-03-01

    A humanised monoclonal antibody, concizumab, that binds with high affinity to the Kunitz-type protease inhibitor (KPI) 2 domain of human tissue factor pathway inhibitor (TFPI) is in clinical development. It promotes coagulation by neutralising the inhibitory function of TFPI and may provide a subcutaneous prophylaxis option for patients with haemophilia. We aimed to study biodistribution and pharmacokinetics (PK) of concizumab. Blockage of cellular TFPI by concizumab was measured by tissue factor/Factor VIIa-mediated Factor X activation on human EA.hy926 cells. Biodistribution of concizumab was analysed in rabbits by immunohistology, and the PK was measured in rabbits and rats. Concizumab bound to cell surface TFPI on EA.hy926 cells and neutralised TFPI inhibition of Factor X activation. The antibody cross-reacted with rabbit TFPI, but not with rat TFPI, allowing for comparative PK studies. PK data in rats described a log-linear profile typical for a non-binding antibody, whereas PK data in rabbits revealed a non-linear, dose-dependent profile, consistent with a target-mediated clearance mechanism. Immunohistology in rabbits during target-saturation showed localisation of the antibody on the endothelium of the microvasculature in several organs. We observed a marked co-localisation with endogenous rabbit TFPI, but a negligible sub-endothelial build-up. Concizumab binds and neutralises the inhibitory effect of cell surface-bound TFPI. The PK profile observed in rabbits is consistent with a TFPI-mediated drug disposition. Double immunofluorescence shows co-localisation of the antibody with TFPI on the endothelium of the microvasculature and points to this TFPI as a putative target involved in the clearance mechanism. Copyright © 2013 Elsevier Ltd. All rights reserved.

  19. Primary structure of a Kunitz-type trypsin inhibitor from Enterolobium contortisiliquum seeds.

    PubMed

    Batista, I F; Oliva, M L; Araujo, M S; Sampaio, M U; Richardson, M; Fritz, H; Sampaio, C A

    1996-03-01

    A trypsin inhibitor was isolated from Enterolobium contortisiliquum seeds. Starting with a saline extract, ECTI (E. contortisiliquum trypsin inhibitor) was purified as a homogeneous protein by acetone precipitation, ion-exchange chromatography (DEAE-Sephadex A-50), gel filtration (Sephadex G-75 and Superose 12) and reversed phase HPLC (mu-Bondapak C-18). The amino acid sequence was determined by automatic degradation and by DABITC/PITC microsequence analysis of the reduced and carboxymethylated protein and also of purified peptides derived from the protein by cleavage with iodosobenzoic acid and by enzymic digestion with trypsin, chymotrypsin and Staphylococcus aureus V8 protease. ECTI contains 174 amino acid residues in two polypeptide chains, an alpha-chain consisting of 134 residues and a beta-chain made up of 40 residues. The inhibitor displays a high degree of sequence identity with other Kunitz-type proteinase inhibitors isolated from the Mimosoideae subfamily. The reactive site was identified (by homology) as the arginine-isoleucine peptide bond at position 64-65. ECTI inhibits trypsin and chymotrypsin in the stoichiometric ratio of 1:1 and also Factor XIIa, plasma kallikrein and plasmin, but not thrombin and Factor Xa.

  20. New Infestin-4 Mutants with Increased Selectivity against Factor XIIa

    PubMed Central

    Vuimo, Tatiana A.; Surov, Stepan S.; Ovsepyan, Ruzanna A.; Korneeva, Vera A.; Vorobiev, Ivan I.; Orlova, Nadezhda A.; Minakhin, Leonid; Kuznedelov, Konstantin; Severinov, Konstantin V.; Ataullakhanov, Fazoil I.; Panteleev, Mikhail A.

    2015-01-01

    Factor XIIa (fXIIa) is a serine protease that triggers the coagulation contact pathway and plays a role in thrombosis. Because it interferes with coagulation testing, the need to inhibit fXIIa exists in many cases. Infestin-4 (Inf4) is a Kazal-type inhibitor of fXIIa. Its specificity for fXIIa can be enhanced by point mutations in the protease-binding loop. We attempted to adapt Inf4 for the selective repression of the contact pathway under various in vitro conditions, e.g., during blood collection and in ‘global’ assays of tissue factor (TF)-dependent coagulation. First, we designed a set of new Inf4 mutants that, in contrast to wt-Inf4, had stabilized canonical conformations during molecular dynamics simulation. Off-target activities against factor Xa (fXa), plasmin, and other coagulation proteases were either reduced or eliminated in these recombinant mutants, as demonstrated by chromogenic assays. Interactions with fXIIa and fXa were also analyzed using protein-protein docking. Next, Mutant B, one of the most potent mutants (its Ki for fXIIa is 0.7 nM) was tested in plasma. At concentrations 5–20 μM, this mutant delayed the contact-activated generation of thrombin, as well as clotting in thromboelastography and thrombodynamics assays. In these assays, Mutant B did not affect coagulation initiated by TF, thus demonstrating sufficient selectivity and its potential practical significance as a reagent for coagulation diagnostics. PMID:26670620

  1. Hemopericardium with tamponade following rivaroxaban administration and its attenuation by CYP3A4 inhibitors

    PubMed Central

    Menendez, Denisse

    2016-01-01

    Novel oral anticoagulants including the factor Xa inhibitor rivaroxaban are important alternatives to warfarin for the prevention of thromboembolic stroke in patients with nonvalvular atrial fibrillation. The pharmacology and metabolism of these agents differ from those of the vitamin K antagonists used over the decades preceding their introduction. We present a case of spontaneous hemopericardium and cardiac tamponade following administration of rivaroxaban. A review of the patient's medications revealed a total of seven agents known to be metabolized through cytochrome P450 3A4 (CYP3A4), the major pathway for rivaroxaban metabolism. While most physicians are familiar with recommendations to monitor renal function in patients prescribed rivaroxaban, we suspect that many fail to evaluate possible interactions with other agents having CYP3A4 inhibitory or inducer activity. PMID:27695181

  2. The Levels of Tissue Factor Pathway Inhibitor in Sepsis Patients Receiving Prophylactic Enoxaparin.

    PubMed

    Al Otair, Hadil A; Abdel Gader, Abdel Galil M; Khurshid, Syed M; Alzeer, Abdulaziz H; Al Momen, Abdul Kareem; Al Shaikh, Mashael; Al Gahtani, Farja; Al Aseri, Zohair A; Abdelrazik, Hossam A H

    2016-06-05

    Amaç: Sepsis sendromuna genellikle kan pıhtılaşma sisteminin aktivasyonu eşlik eder. İlk çalışmalar ana doğal 3 antikoagülan olan antitrombin, protein C ve protein S eksikliği bulmuştur. Bununla birlikte, bu inhibitörlerin hiç biri doku faktörü bloke etmez, sepsis sırasındaki koagülasyon tetiklenişi özelllikle doku faktör yolak inhibitörü (DFYİ) ile kontrol edilir. Bu çalışmanın amacı sepsis sırasındaki doğal antikoagülan ve özellikle DFYİ düzeyi dalgalanmalarını karakterize etmek ve bunların düşük moleküler ağırlıklı heaprin enoksaparinin antikoagülan eylemi ile ilişkilerini öğrenmekti. Gereç ve Yöntemler: Ardışık 51 sepsis hastası çalışmaya alındı. Taban (0 saat) ve enoksaparin verimesinden 4, 12, 24 saat sonra kan örnekleri alındı. Aşağıdaki deneyler ticari kitleri kullanılarak yapılmıştır; parsiyel tromboplastin zamanı, protrombin zamanı, trombin zamanı, toplam ve serbest DFYİ, protein C ve protein S, antitrombin, fibrinojen, ve aktif anti-faktör Xa. Bulgular: Enoksaparin uygulamadan önce ptorombin zamanı ve aktif parsiyel protrombin zamanında önemli uzama vardı. Bu durum sonraki 3 örneklemde de devam etti. Çalışma boyunca antitrombin, protein C, toplam ve serbest protein S seviyeleri değerlerinde kontrollere göre belirgin bir azalma oldu. Buna karşılık, hem toplam hem de serbest plazma DFYİ değerleri belirgin biçimde yükseldi ve enoksaparin tedavisinden sonra arttı. Anti faktör Xa düzeyleri terapötik aralık içindeydi. Vefat eden ve sağ kalan hastalar arasında DFYİ düzeyi açısından fark yoktu. Sonuç: Sepsis, endotel hücrelerinden belirgin DFYİ salınımı ile tetiklenir. Bu, enoksaparin uygulmasını takiben kalıcı olmuş ve daha da artmıştır. Bunun aksine, doğal koagülasyon inhibitörleri antitrombin, protein C ve protein S’nin belirgin tüketimi vardı. Bu sonuçlar, tedavi amaçlı rekombinant DFYİ kullanımının sepsis ilişkili koag

  3. Effects of tumor necrosis factor α inhibitors extend beyond psoriasis: insulin sensitivity in psoriasis patients with type 2 diabetes mellitus.

    PubMed

    Al-Mutairi, Nawaf; Shabaan, Dalia

    2016-03-01

    Psoriasis is a chronic inflammatory disease that has been associated with an increased incidence of insulin resistance and diabetes mellitus (DM). Tumor necrosis factor (TNF) α inhibitors and IL-6 blockers, which are routinely used for the treatment of psoriasis, have been positively associated with insulin sensitivity. The aim of this study was to assess the effects of treatment with TNF-α inhibitors on insulin sensitivity in psoriatic patients with type 2 DM. This study confirms a beneficial effect of anti-TNF-α agents on insulin resistance and insulin sensitivity in psoriasis patients with type 2 DM.

  4. Addressing epidermal growth factor receptor tyrosine kinase inhibitor resistance in non-small cell lung cancer.

    PubMed

    Noda, Shoko; Kanda, Shintaro

    2016-01-01

    Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) have significantly improved the survival of patients with advanced non-small cell lung cancer (NSCLC) harboring EGFR activating mutations. However, nearly all EGFR-mutant NSCLC tumors eventually acquire resistance to the currently used EGFR-TKIs and subsequently progress clinically. Acquired resistance to EGFR-TKIs is thus a huge issue in the treatment of EGFR-mutant NSCLC at present. On one hand, T790M second-site mutation has been recognized as a key mechanism of EGFR-TKI resistance, and third generation EGFR-TKIs such as osimertinib and rociletinib have been developed to overcome tumor cells harboring the T790M mutation. On the other hand, combination with cytotoxic chemotherapy is also expected as another strategy for preventing the acquired resistance to current EGFR-TKIs and prolonging the survival benefits by EGFR-TKIs. Here, we review updated strategies for preventing or overcoming acquired resistance to EGFR-TKIs.

  5. A-62176, a potent topoisomerase inhibitor, inhibits the expression of human epidermal growth factor receptor 2.

    PubMed

    Kim, Hye-Lin; Jeon, Kyung-Hwa; Jun, Kyu-Yeon; Choi, Yongmun; Kim, Dae-Kee; Na, Younghwa; Kwon, Youngjoo

    2012-12-01

    HER2 overexpression is observed in ∼6-35% of all gastric cancers, while co-amplification of topoisomerase IIα occurs in ∼32-90% of all cancers with HER2 amplification. The present study reports that HER2 expression is down-regulated by A-62176, a fluoroquinophenoxazine derivative that we previously demonstrated to inhibit topoisomerase I and IIα. The results suggest that A-62176 inhibits the interaction between the ESX, an ets transcription factor, and its co-activator Sur2, leading to the attenuation of HER2-mediated phosphorylation of MAPK/Akt. A-62176 arrests the cell cycle in the G1 phase via the down-regulation of cyclin D1 and the up-regulation of p27(Kip1) in NCI-N87 gastric cancer cells. The combination of A-62176 with doxorubicin provides a strong synergistic activity. We propose that A-62176 is a dual inhibitor that impairs the expression of HER2 and restrains the activity of topoisomerase IIα. Our results may lead to the rational design of anticancer molecules targeting a subgroup of gastric cancer cells overexpressing both HER2 and topoisomerase IIα.

  6. Monoclonal antibodies in rheumatoid arthritis: comparative effectiveness of tocilizumab with tumor necrosis factor inhibitors

    PubMed Central

    Tanaka, Toshio; Hishitani, Yoshihiro; Ogata, Atsushi

    2014-01-01

    Rheumatoid arthritis (RA) is a chronic inflammatory disease characterized by persistent joint inflammation, systemic inflammation, and immunological abnormalities. Because cytokines such as tumor necrosis factor (TNF)-α and interleukin (IL)-6 play a major role in the development of RA, their targeting could constitute a reasonable novel therapeutic strategy for treating RA. Indeed, worldwide clinical trials of TNF inhibiting biologic disease modifying antirheumatic drugs (bDMARDs) including infliximab, adalimumab, golimumab, certolizumab pegol, and etanercept as well as the humanized anti-human IL-6 receptor antibody, tocilizumab, have demonstrated outstanding clinical efficacy and tolerable safety profiles, resulting in worldwide approval for using these bDMARDs to treat moderate to severe active RA in patients with an inadequate response to synthetic disease modifying antirheumatic drugs (sDMARDs). Although bDMARDs have elicited to a paradigm shift in the treatment of RA due to the prominent efficacy that had not been previously achieved by sDMARDs, a substantial percentage of patients failed primary or secondary responses to bDMARD therapy. Because RA is a heterogeneous disease in which TNF-α and IL-6 play overlapping but distinct pathological roles, further studies are required to determine the best use of TNF inhibitors and tocilizumab in individual RA patients. PMID:24741293

  7. Vascular endothelial growth factor inhibitor use and treatment approach for choroidal neovascularization secondary to pathologic myopia.

    PubMed

    Pakzad-Vaezi, Kaivon; Mehta, Hemal; Mammo, Zaid; Tufail, Adnan

    2016-07-01

    Myopic choroidal neovascularization (CNV) is the most common cause of CNV in those under 50 years of age. It is a significant cause of visual loss in those with pathologic myopia. The current standard of care involves therapy with intravitreal inhibitors of vascular endothelial growth factor (VEGF). The epidemiology of myopia, high myopia, pathologic myopia, and myopic CNV is reviewed, along with a brief discussion of historical treatments. The pharmacology of the three most commonly used anti-VEGF agents is discussed, with an emphasis on the licensed drugs, ranibizumab and aflibercept. A comprehensive clinical approach to diagnosis and treatment of myopic CNV is presented. The current standard of care for myopic CNV is intravitreal inhibition of VEGF, with ranibizumab and aflibercept licensed for intraocular use. The diagnosis, OCT features of disease activity and retreatment algorithm for myopic CNV is different from wet age-related macular degeneration. In the long-term, myopic CNV may be associated with gradual, irreversible visual loss due to progressive chorioretinal atrophy, for which there is currently no treatment.

  8. Third-generation epidermal growth factor receptor tyrosine kinase inhibitors in advanced nonsmall cell lung cancer.

    PubMed

    Pirker, Robert

    2016-03-01

    Patients with epidermal growth factor receptor (EGFR) mutation-positive nonsmall cell lung cancer (NSCLC) develop resistance during therapy with EGFR tyrosine kinase inhibitors (TKIs). In about half of the patients, this resistance is because of the emergence of the T790M mutation. Third-generation TKIs are active against EGFR-activating mutations and the T790M resistance mutation and have only limited efficacy against wild-type EGFR. Here we review the current status of the clinical development of these novel TKIs. Third-generation TKIs in clinical development include osimertinib, rociletinib, and HM61713. Osimertinib and rociletinib have shown clinical efficacy in phase I/II trials in patients who had acquired resistance to first- or second-generation TKIs. Both TKIs are currently further evaluated in phase III trials as first-line or second-line therapy in patients with advanced EGFR mutation-positive NSCLC. HM61713 is in early clinical development. Third-generation EGFR TKIs have shown activity in patients with acquired resistance to first- and second-generation EGFR TKIs and may further improve clinical outcome in patients with advanced EGFR mutation-positive NSCLC.

  9. Regulation of epidermal growth factor receptor signalling by inducible feedback inhibitors.

    PubMed

    Segatto, Oreste; Anastasi, Sergio; Alemà, Stefano

    2011-06-01

    Signalling by the epidermal growth factor receptor (EGFR) controls morphogenesis and/or homeostasis of several tissues from worms to mammals. The correct execution of these programmes requires the generation of EGFR signals of appropriate strength and duration. This is obtained through a complex circuitry of positive and negative feedback regulation. Feedback inhibitory mechanisms restrain EGFR activity in time and space, which is key to ensuring that receptor outputs are commensurate to the cell and tissue needs. Here, we focus on the emerging field of inducible negative feedback regulation of the EGFR in mammals. In mammalian cells, four EGFR inducible feedback inhibitors (IFIs), namely LRIG1, RALT (also known as MIG6 and ERRFI1), SOCS4 and SOCS5, have been discovered recently. EGFR IFIs are expressed de novo in the context of early or delayed transcriptional responses triggered by EGFR activation. They all bind to the EGFR and suppress receptor signalling through several mechanisms, including catalytic inhibition and receptor downregulation. Here, we review the mechanistic basis of IFI signalling and rationalise the function of IFIs in light of gene-knockout studies that assign LRIG1 and RALT an essential role in restricting cell proliferation. Finally, we discuss how IFIs might participate in system control of EGFR signalling and highlight the emerging roles for IFIs in the suppression of EGFR-driven tumorigenesis.

  10. Tumor necrosis factorinhibitor therapy and fetal risk: A systematic literature review

    PubMed Central

    Marchioni, Renée M; Lichtenstein, Gary R

    2013-01-01

    Tumor necrosis factorinhibitors (anti-TNFs) are effective in the treatment of inflammatory bowel disease (IBD) recalcitrant to conventional medical therapy. As the peak incidence of IBD overlaps with the prime reproductive years, it is crucial to establish pharmacologic regimens for women of childbearing age that achieve effective disease control without posing significant fetal harm. A systematic literature review was performed to identify all human studies with birth outcomes data after maternal exposure to infliximab, adalimumab, or certolizumab pegol within 3 mo of conception or during any trimester of pregnancy. Live births, spontaneous abortions or stillbirths, preterm or premature births, low birth weight or small for gestational age infants, and congenital abnormalities were recorded. Fifty selected references identified 472 pregnancy exposures. The subsequent review includes general information regarding anti-TNF therapy in pregnancy followed by a summary of our findings. The benefits of biologic modalities in optimizing disease control during pregnancy must be weighed against the potential toxicity of drug exposure on the developing fetus. Although promising overall, there is insufficient evidence to prove absolute safety for use of anti-TNFs during pregnancy given the limitations of available data and lack of controlled trials. PMID:23674866

  11. Epidermal growth factor receptor inhibitor protects against abdominal aortic aneurysm in a mouse model.

    PubMed

    Obama, Takashi; Tsuji, Toshiyuki; Kobayashi, Tomonori; Fukuda, Yamato; Takayanagi, Takehiko; Taro, Yoshinori; Kawai, Tatsuo; Forrester, Steven J; Elliott, Katherine J; Choi, Eric; Daugherty, Alan; Rizzo, Victor; Eguchi, Satoru

    2015-05-01

    Angiotensin II (Ang II) has been implicated in the development of abdominal aortic aneurysm (AAA). In vascular smooth muscle cells (VSMC), Ang II activates epidermal growth factor receptor (EGFR) mediating growth promotion. We hypothesized that inhibition of EGFR prevents Ang II-dependent AAA. C57BL/6 mice were co-treated with Ang II and β-aminopropionitrile (BAPN) to induce AAA with or without treatment with EGFR inhibitor, erlotinib. Without erlotinib, 64.3% of mice were dead due to aortic rupture. All surviving mice had AAA associated with EGFR activation. Erlotinib-treated mice did not die and developed far fewer AAA. The maximum diameters of abdominal aortas were significantly shorter with erlotinib treatment. In contrast, both erlotinib-treated and non-treated mice developed hypertension. The erlotinib treatment of abdominal aorta was associated with lack of EGFR activation, endoplasmic reticulum (ER) stress, oxidative stress, interleukin-6 induction and matrix deposition. EGFR activation in AAA was also observed in humans. In conclusion, EGFR inhibition appears to protect mice from AAA formation induced by Ang II plus BAPN. The mechanism seems to involve suppression of vascular EGFR and ER stress.

  12. Energetic factors determining the binding of type I inhibitors to c-Met kinase: experimental studies and quantum mechanical calculations

    PubMed Central

    Yu, Zhe; Ma, Yu-chi; Ai, Jing; Chen, Dan-qi; Zhao, Dong-mei; Wang, Xin; Chen, Yue-lei; Geng, Mei-yu; Xiong, Bing; Cheng, Mao-sheng; Shen, Jing-kang

    2013-01-01

    Aim: To decipher the molecular interactions between c-Met and its type I inhibitors and to facilitate the design of novel c-Met inhibitors. Methods: Based on the prototype model inhibitor 1, four ligands with subtle differences in the fused aromatic rings were synthesized. Quantum chemistry was employed to calculate the binding free energy for each ligand. Symmetry-adapted perturbation theory (SAPT) was used to decompose the binding energy into several fundamental forces to elucidate the determinant factors. Results: Binding free energies calculated from quantum chemistry were correlated well with experimental data. SAPT calculations showed that the predominant driving force for binding was derived from a sandwich π–π interaction with Tyr-1230. Arg-1208 was the differentiating factor, interacting with the 6-position of the fused aromatic ring system through the backbone carbonyl with a force pattern similar to hydrogen bonding. Therefore, a hydrogen atom must be attached at the 6-position, and changing the carbon atom to nitrogen caused unfavorable electrostatic interactions. Conclusion: The theoretical studies have elucidated the determinant factors involved in the binding of type I inhibitors to c-Met. PMID:24056705

  13. Energetic factors determining the binding of type I inhibitors to c-Met kinase: experimental studies and quantum mechanical calculations.

    PubMed

    Yu, Zhe; Ma, Yu-chi; Ai, Jing; Chen, Dan-qi; Zhao, Dong-mei; Wang, Xin; Chen, Yue-lei; Geng, Mei-yu; Xiong, Bing; Cheng, Mao-sheng; Shen, Jing-Kang

    2013-11-01

    To decipher the molecular interactions between c-Met and its type I inhibitors and to facilitate the design of novel c-Met inhibitors. Based on the prototype model inhibitor 1, four ligands with subtle differences in the fused aromatic rings were synthesized. Quantum chemistry was employed to calculate the binding free energy for each ligand. Symmetry-adapted perturbation theory (SAPT) was used to decompose the binding energy into several fundamental forces to elucidate the determinant factors. Binding free energies calculated from quantum chemistry were correlated well with experimental data. SAPT calculations showed that the predominant driving force for binding was derived from a sandwich π-π interaction with Tyr-1230. Arg-1208 was the differentiating factor, interacting with the 6-position of the fused aromatic ring system through the backbone carbonyl with a force pattern similar to hydrogen bonding. Therefore, a hydrogen atom must be attached at the 6-position, and changing the carbon atom to nitrogen caused unfavorable electrostatic interactions. The theoretical studies have elucidated the determinant factors involved in the binding of type I inhibitors to c-Met.

  14. Nitric oxide synthase inhibitors attenuate transforming-growth-factor-beta 1-stimulated capillary organization in vitro.

    PubMed Central

    Papapetropoulos, A.; Desai, K. M.; Rudic, R. D.; Mayer, B.; Zhang, R.; Ruiz-Torres, M. P.; García-Cardeña, G.; Madri, J. A.; Sessa, W. C.

    1997-01-01

    Angiogenesis is a complex process involving endothelial cell (EC) proliferation, migration, differentiation, and organization into patent capillary networks. Nitric oxide (NO), an EC mediator, has been reported to be antigenic as well as proangiogenic in different models of in vivo angiogenesis. Our aim was to investigate the role of NO in capillary organization using rat microvascular ECs (RFCs) grown in three-dimensional (3D) collagen gels. RFCs placed in 3D cultures exhibited extensive tube formation in the presence of transforming growth factor-beta 1. Addition of the NO synthase (NOS) inhibitors L-nitro-arginine methylester (L-NAME, 1 mmol/L) or L-monomethyl-nitro-l-arginine (1 mmol/L) inhibited tube formation and the accumulation of nitrite in the media by approximately 50%. Incubation of the 3D cultures with excess L-arginine reversed the inhibitory effect of L-NAME on tube formation. In contrast to the results obtained in 3D cultures, inhibition of NO synthesis by L-NAME did not influence RFC proliferation in two-dimensional (2D) cultures or antagonize the ability of transforming growth factor-beta 1 to suppress EC proliferation in 2D cultures. Reverse transcriptase-polymerase chain reaction revealed the constitutive expression of all three NOS isoforms, neuronal, inducible, and endothelial NOSs, in 2D and 3D cultures. Moreover, Western blot analysis demonstrated the presence of immunoreactive protein for all NOS isoforms in 3D cultures of RFCs. In addition, in the face of NOS blockade, co-treatment with the NO donor sodium nitroprusside or the stable analog of cGMP, 8-bromo-cGMP, restored capillary tube formation. Thus, the autocrine production of NO and the activation of soluble guanylate cyclase are necessary events in the process of differentiation and in vitro capillary tube organization of RFCs. Images Figure 2 Figure 4 Figure 5 PMID:9137106

  15. Parents' information needs in tumor necrosis factorinhibitor treatment decisions.

    PubMed

    Lipstein, Ellen A; Lovell, Daniel J; Denson, Lee A; Moser, David W; Saeed, Shehzad A; Dodds, Cassandra M; Britto, Maria T

    2013-03-01

    The aim of the study was to describe parents' experiences and the information used when making decisions about tumor necrosis factorinhibitor (TNFαi) treatment. We interviewed parents of children with Crohn disease (CD) or juvenile idiopathic arthritis who had experience deciding about TNFαi treatment. Interview questions focused on information used to make decisions and factors that influenced decision making. We used thematic analysis for all coding and analysis. Coding structure was developed by a multidisciplinary team review of the initial interviews. Two coders then coded the remaining interviews, compared coding, and resolved disagreements through discussion. Data were analyzed by thematic grouping and then compared between diseases. We interviewed 35 parents. For nearly all parents the decision about TNFαi treatment was the most challenging medical decision they had made; however, parents of children with CD experienced more stress and anxiety than did other parents. Both groups of parents sought information from multiple sources including health care providers, the Internet, and social contacts. They looked for information related to treatment effectiveness, adverse effects, and other individuals' treatment experiences. In CD, information was used to help make the decision, whereas in juvenile idiopathic arthritis it was used to confirm the decision. The decision-making experience, and associated information seeking, leaves some parents with long-lasting concerns and worry about TNFαi treatment. Providing parents with structured decision-making support may lead to more effective and efficient decision making, decreased psychosocial distress, and, ultimately, improved outcomes for their children.

  16. Management of dermatologic toxicities associated with monoclonal antibody epidermal growth factor receptor inhibitors: A case review

    PubMed Central

    Leporini, Christian; Saullo, Francesca; Filippelli, Gianfranco; Sorrentino, Antonio; Lucia, Maria; Perri, Gino; Gattuta, Gaetana La; Infusino, Stefania; Toscano, Rosa; Dima, Gianluca; Olivito, Virginia; Paletta, Laura; Bottoni, Ugo; De Sarro, Giovambattista

    2013-01-01

    Introduction: The epidermal growth factor receptor inhibitors (EGFRIs), cetuximab and panitumumab, represent an effective treatment option for patients affected by metastatic colorectal cancer (mCRC); furthermore, they are relatively devoid of systemic toxicities, which are commonly observed with standard cytotoxic chemotherapy. However, the majority of patients treated with these monoclonal antibodies (mAbs), will experience dermatologic toxicities, most notably the papulopustular skin rash, which can impact quality-of-life and affect adherence to therapy. This paper reviews the most recent practices in the management of skin rash related to anti-epidermal growth factor receptor (EGFR) mAbs, cetuximab and panitumumab, in the treatment of mCRC. Materials and Methods: We reviewed relevant literature regarding dermatologic toxicities associated with anti-EGFR mAbs in order to give important indications about prevention and reactive treatment of skin rash. Results: Two case reports were presented to show how skin rash could hamper mAb EGFRIs use in clinical practice, underscoring the need of implementing a comprehensive management strategy of skin toxicity in order to promote patients’ compliance with anti-EGFR therapy and maintain quality-of-life. Based on randomized data, recent guidelines established by the Multinational Association for Supportive Care in Cancer Skin Toxicity Study Group suggest that prophylactic use of oral doxycycline or minocycline reduces the risk and severity of skin rash, improving clinical outcomes. Conclusions: At the start of treatment with cetuximab and panitumumab, the proper patient education about the skin rash associated with these mAbs and the implementation of a pre-emptive, comprehensive skin toxicity program significantly contribute to improve adherence to therapy, optimize anti-EGFR therapy and maintain quality-of-life. PMID:24347989

  17. The M358R variant of α(1)-proteinase inhibitor inhibits coagulation factor VIIa.

    PubMed

    Sheffield, William P; Bhakta, Varsha

    2016-02-12

    The naturally occurring M358R mutation of the plasma serpin α1-proteinase inhibitor (API) changes both its cleavable reactive centre bond to Arg-Ser and the efficacy with which it inhibits different proteases, reducing the rate of inhibition of neutrophil elastase, and enhancing that of thrombin, factor XIa, and kallikrein, by several orders of magnitude. Although another plasma serpin with an Arg-Ser reactive centre, antithrombin (AT), has been shown to inhibit factor VIIa (FVIIa), no published data are available with respect to FVIIa inhibition by API M358R. Recombinant bacterially-expressed API M358R and plasma-derived AT were therefore compared using gel-based and kinetic assays of FVIIa integrity and activity. Under pseudo-first order conditions of excess serpin over protease, both AT and API M358R formed denaturation-resistant inhibitory complexes with FVIIa in reactions accelerated by TF; AT, but not API M358R, also required heparin for maximal activity. The second order rate constant for heparin-independent API M358R-mediated FVIIa inhibition was determined to be 7.8 ± 0.8 × 10(2) M(-1)sec(-1). We conclude that API M358R inhibits FVIIa by forming inhibitory complexes of the serpin type more rapidly than AT in the absence of heparin. The likely 20-fold excess of API M358R over AT in patient plasma during inflammation raises the possibility that it could contribute to the hemorrhagic tendencies manifested by rare individuals expressing this mutant serpin.

  18. Predictive Factors of Response to Proton Pump Inhibitors in Korean Patients With Gastroesophageal Reflux Disease

    PubMed Central

    Kim, Sung Eun; Kim, Nayoung; Oh, Sooyeon; Kim, Hee Man; Park, Moo In; Lee, Dong Ho; Jung, Hyun Chae

    2015-01-01

    Background/Aims Proton pump inhibitors (PPIs) are widely used in the treatment of gastroesophageal reflux disease (GERD). However, some patients fail to respond to PPI therapy. We investigated the efficacy of response to PPI therapy in patients with GERD symptoms. Methods A total of 179 subjects with GERD symptoms were prospectively enrolled and diagnosed with non-erosive reflux disease (NERD, n = 100) and erosive reflux disease (n = 79) by gastroscopy and Bernstein test and/or 24-hour esophageal pH testing. Subjects then received a standard dose of daily PPI therapy for at least 4 weeks. PPI therapy response was evaluated using questionnaires including questions about demographics, GERD symptoms, GERD impact scale, Epworth sleepiness scale, Pittsburgh sleep quality index (PSQI), hospital anxiety and depression scale, and abbreviated version of the World Health Organization quality of life scale. Results The rates of complete (≥ 80%), satisfactory (≥ 50%), partial (< 50%), and refractory response in the 179 participants were 41.3%, 30.2%, 18.4%, and 10.1%, respectively. Thus, overall response rate (complete and satisfactory responses) was 71.5%. Multivariate analysis showed body mass index < 23 kg/m2 (OR, 2.20; 95% CI, 1.12–4.34), higher total PSQI score (OR, 1.20; 95% CI, 1.05–1.35), history of psychotherapy or neuropsychiatric medication (OR, 2.44; 95% CI, 1.23–4.85), and NERD (OR, 3.30; 95% CI, 1.54–7.11) were associated with poor response to PPI therapy. Conclusions Psychological factors, sleep dysfunction, body mass index < 23 kg/m2, and NERD seem to be the major factors that lead to a poor response to PPI treatment in patients with GERD symptoms. PMID:25537676

  19. Factors associated with residual gastroesophageal reflux disease symptoms in patients receiving proton pump inhibitor maintenance therapy

    PubMed Central

    Kawara, Fumiaki; Fujita, Tsuyoshi; Morita, Yoshinori; Uda, Atsushi; Masuda, Atsuhiro; Saito, Masaya; Ooi, Makoto; Ishida, Tsukasa; Kondo, Yasuyuki; Yoshida, Shiei; Okuno, Tatsuya; Yano, Yoshihiko; Yoshida, Masaru; Kutsumi, Hiromu; Hayakumo, Takanobu; Yamashita, Kazuhiko; Hirano, Takeshi; Hirai, Midori; Azuma, Takeshi

    2017-01-01

    AIM To elucidate the factors associated with residual gastroesophageal reflux disease (GERD) symptoms in patients receiving proton pump inhibitor (PPI) maintenance therapy in clinical practice. METHODS The study included 39 GERD patients receiving maintenance PPI therapy. Residual symptoms were assessed using the Frequency Scale for Symptoms of GERD (FSSG) questionnaire and the Gastrointestinal Symptom Rating Scale (GSRS). The relationships between the FSSG score and patient background factors, including the CYP2C19 genotype, were analyzed. RESULTS The FSSG scores ranged from 1 to 28 points (median score: 7.5 points), and 19 patients (48.7%) had a score of 8 points or more. The patients’ GSRS scores were significantly correlated with their FSSG scores (correlation coefficient = 0.47, P < 0.005). In erosive esophagitis patients, the FSSG scores of the CYP2C19 rapid metabolizers (RMs) were significantly higher than the scores of the poor metabolizers and intermediate metabolizers (total scores: 16.7 ± 8.6 vs 7.8 ± 5.4, P < 0.05; acid reflux-related symptom scores: 12 ± 1.9 vs 2.5 ± 0.8, P < 0.005). In contrast, the FSSG scores of the CYP2C19 RMs in the non-erosive reflux disease patients were significantly lower than those of the other patients (total scores: 5.5 ± 1.0 vs 11.8 ± 6.3, P < 0.05; dysmotility symptom-related scores: 1.0 ± 0.4 vs 6.0 ± 0.8, P < 0.01). CONCLUSION Approximately half of the GERD patients receiving maintenance PPI therapy had residual symptoms associated with a lower quality of life, and the CYP2C19 genotype appeared to be associated with these residual symptoms. PMID:28373773

  20. Soluble biomarkers associated with response to treatment with tumor necrosis factor inhibitors in psoriatic arthritis.

    PubMed

    Chandran, Vinod; Shen, Hua; Pollock, Remy A; Pellett, Fawnda J; Carty, Adele; Cook, Richard J; Gladman, Dafna D

    2013-06-01

    To identify soluble biomarkers associated with response to therapy with tumor necrosis factor inhibitors (TNFi) in patients with psoriatic arthritis (PsA). The study was conducted at a PsA clinic where patients are assessed every 6 months, and serum samples are collected and stored once a year at the time of clinical assessment. Forty patients with active PsA who gave serum samples prior to treatment with TNFi and after at least 3 months of therapy were identified. Patients were classified as TNFi responders if tender joint count was < 3, swollen joint count was 0, and Psoriasis Area and Severity Index score was < 4 at the time the second sample was collected. The following biomarkers were tested by ELISA: TNF superfamily 14, matrix metalloprotease-3 (MMP-3), receptor activator of nuclear factor kappa-B ligand, osteoprotegerin, cartilage oligomeric matrix protein (COMP), CPII, C2C and C1-2C, CS-846, and highly sensitive C-reactive protein. Paired t tests and logistic regression was used for statistical analyses. After a mean treatment duration of 11 months with TNFi (etanercept 28 patients, adalimumab 6, golimumab 4, infliximab 2), 29 patients were classified as TNFi responders. Baseline level of MMP-3 was independently associated with responder status (OR 1.067 for each 1-unit increase, p = 0.045). A reduction in MMP-3 levels with therapy increased the odds of achieving response (OR 1.213 for each 1-unit change, p = 0.030), whereas a reduction in COMP decreased the odds (OR 0.587, for each 100-unit increase, p = 0.039). None of the other biomarkers was associated with response. Baseline as well as reduction in serum MMP-3 and increase in serum COMP are independently associated with response to TNFi therapy in patients with PsA.

  1. Effect of Tumor Necrosis Factor Inhibitor Therapy on Osteoclasts Precursors in Ankylosing Spondylitis

    PubMed Central

    Caetano-Lopes, Joana; Vieira-Sousa, Elsa; Campanilho-Marques, Raquel; Ponte, Cristina; Canhão, Helena; Ainola, Mari; Fonseca, João E.

    2015-01-01

    Introduction Ankylosing Spondylitis (AS) is characterized by excessive local bone formation and concomitant systemic bone loss. Tumor necrosis factor (TNF) plays a central role in the inflammation of axial skeleton and enthesis of AS patients. Despite reduction of inflammation and systemic bone loss, AS patients treated with TNF inhibitors (TNFi) have ongoing local bone formation. The aim of this study was to assess the effect of TNFi in the differentiation and activity of osteoclasts (OC) in AS patients. Methods 13 AS patients treated with TNFi were analyzed at baseline and after a minimum follow-up period of 6 months. 25 healthy donors were recruited as controls. Blood samples were collected to assess receptor activator of nuclear factor kappa-B ligand (RANKL) surface expression on circulating leukocytes and frequency and phenotype of monocyte subpopulations. Quantification of serum levels of bone turnover markers and cytokines, in vitro OC differentiation assay and qRT-PCR for OC specific genes were performed. Results RANKL+ circulating lymphocytes (B and T cells) and IL-17A, IL-23 and TGF-β levels were decreased after TNFi treatment. We found no differences in the frequency of the different monocyte subpopulations, however, we found decreased expression of CCR2 and increased expression of CD62L after TNFi treatment. OC number was reduced in patients at baseline when compared to controls. OC specific gene expression was reduced in circulating OC precursors after TNFi treatment. However, when cultured in OC differentiating conditions, OC precursors from AS TNFi-treated patients showed increased activity as compared to baseline. Conclusion In AS patients, TNFi treatment reduces systemic pro osteoclastogenic stimuli. However, OC precursors from AS patients exposed to TNFi therapy have increased in vitro activity in response to osteoclastogenic stimuli. PMID:26674064

  2. Structure based design of 4-(3-aminomethylphenyl)piperidinyl-1-amides: novel, potent, selective, and orally bioavailable inhibitors of betaII tryptase.

    PubMed

    Levell, Julian; Astles, Peter; Eastwood, Paul; Cairns, Jennifer; Houille, Olivier; Aldous, Suzanne; Merriman, Gregory; Whiteley, Brian; Pribish, James; Czekaj, Mark; Liang, Guyan; Maignan, Sebastien; Guilloteau, Jean-Pierre; Dupuy, Alain; Davidson, Jane; Harrison, Trevor; Morley, Andrew; Watson, Simon; Fenton, Garry; McCarthy, Clive; Romano, Joseph; Mathew, Rose; Engers, Darren; Gardyan, Michael; Sides, Keith; Kwong, Jennifer; Tsay, Joseph; Rebello, Sam; Shen, Liduo; Wang, Jie; Luo, Yongyi; Giardino, Odessa; Lim, Heng-Keang; Smith, Keith; Pauls, Henry

    2005-04-15

    Tryptase is a serine protease found almost exclusively in mast cells. It has trypsin-like specificity, favoring cleavage of substrates with an arginine (or lysine) at the P1 position, and has optimal catalytic activity at neutral pH. Current evidence suggests tryptase beta is the most important form released during mast cell activation in allergic diseases. It is shown to have numerous pro-inflammatory cellular activities in vitro, and in animal models tryptase provokes broncho-constriction and induces a cellular inflammatory infiltrate characteristic of human asthma. Screening of in-house inhibitors of factor Xa (a closely related serine protease) identified beta-amidoester benzamidines as potent inhibitors of recombinant human betaII tryptase. X-ray structure driven template modification and exchange of the benzamidine to optimize potency and pharmacokinetic properties gave selective, potent and orally bioavailable 4-(3-aminomethyl phenyl)piperidinyl-1-amides.

  3. [Pyramiding of senescence-inhibition IPT gene and Xa23 for resistance to bacterial blight in rice (Oryza sativa L.)].

    PubMed

    He, Guang-Ming; Sun, Chuan-Qing; Fu, Yong-Cai; Fu, Qiang; Zhao, Kai-Jun; Wang, Chun-Lian; Zhang, Qi; Ling, Zhong-Zhuan; Wang, Xiang-Kun

    2004-08-01

    Transgenic lines (GC-1) carrying a senescence-inhibition cheimeric gene, IPT (isopentenyl transferase) gene, CBB23, a isogenic lines carrying Xa23 gene for resistance to bacterial blight, and Hexi15, a commercial cultivar showing high resistance to blast disease, were used as donors to pyramid IPT gene and Xa23 by marker-assisted selection (MAS). Seventeen BC1F1 plants pyramiding Xa23 gene and IPT genes were obtained from three multi-cross combinations. Then, the plants carrying Xa23 and IPT genes were crossed with parental lines of two-line hybrid rice, such as 9311, E32, Pei' ai 64S and W9834S. The progenies were backcrossed the acceptor parents. A total of 17 plants carrying Xa23 and IPT genes were detected by PCR, disease resistance identification and analysis of CTK contents of in the four combinations of "(9311///Hexi15/CBB23// GC-1) x 9311", "(E32///Hexi15/CBB23//GC-1) x E32", "(Pei'ai 64S///Hexi15/CBB23//GC-1) x Pei' ai 64S" and "(GC-1/CBB23//W9834S/Hexi15) x W9834S". These plants showed resistance to blast disease by inoculating test using 21 the lines of Pyricularia grisea from Northern China. Six plants of BC2F1 pyramiding Xa23 and IPT genes were further obtained in the combinations of "[(9311///Hexi15/CBB23//GC-1) x 9311] x 9311", "[(E32///Hexi15/CBB23//GC-1) x E32] x E32". After backcrossed and self-crossed 1 approximately 2nd, the plants pyramiding Xa23 and IPT genes can be used in the program of hybrid rice breeding.

  4. Hemophilia as a defect of the tissue factor pathway of blood coagulation: Effect of factors VIII and IX on factor X activation in a continuous-flow reactor

    SciTech Connect

    Repke, D.; Gemmell, C.H.; Guha, A.; Turitto, V.T.; Nemerson, Y. ); Broze, G.J. Jr. )

    1990-10-01

    The effect of factors VIII and IX on the ability of the tissue factor-factor VIIa complex to activate factor X was studied in a continuous-flow tubular enzyme reactor. Tissue factor immobilized in a phospholipid bilayer on the inner surface of the tube was exposed to a perfusate containing factors VIIa, VIII, IX, and X flowing at a wall shear rate of 57, 300, or 1130 sec{sup {minus}1}. The addition of factors VIII and IX at their respective plasma concentrations resulted in a further 2{endash}-to 3{endash}fold increase. The direct activation of factor X by tissue factor-factor VIIa could be virtually eliminated by the lipoprotein-associated coagulation inhibitor. These results suggest that the tissue factor pathway, mediated through factors VIII and IX, produces significant levels of factor Xa even in the presence of an inhibitor of the tissue factor-factor VIIa complex; moreover, the activation is dependent on local shear conditions. These findings are consistent both with a model of blood coagulation in which initiation of the system results from tissue factor and with the bleeding observed in hemophilia.

  5. Limit of detection and threshold for positivity of the Centers for Disease Control and Prevention assay for factor VIII inhibitors.

    PubMed

    Miller, C H; Boylan, B; Shapiro, A D; Lentz, S R; Wicklund, B M

    2017-08-10

    Essentials Immunologic methods detect factor VIII (FVIII) antibodies in some inhibitor-negative specimens. Specimens were tested by modified Nijmegen-Bethesda assay (NBA) and fluorescence immunoassay. The NBA with preanalytical heat inactivation detects FVIII inhibitors down to 0.2 NBU. IgG4 frequency validates the established threshold for positivity of ≥ 0.5 NBU for this NBA. Background The Bethesda assay for measurement of factor VIII inhibitors called for quantification of positive inhibitors by using dilutions producing 25-75% residual activity (RA), corresponding to 0.4-2.0 Bethesda units, with the use of 'more sensitive methods' for samples with RA closer to 100% being recommended. The Nijmegen modification (Nijmegen-Bethesda assay [NBA]) changed the reagents used but not these calculations. Some specimens negative by the NBA have been shown to have FVIII antibodies detectable with sensitive immunologic methods. Objective To examine the performance at very low inhibitor titers of the Centers for Disease Control and Prevention (CDC)-modified NBA (CDC-NBA), which includes preanalytic heat inactivation to liberate bound anti-FVIII antibodies. Methods Specimens with known inhibitors were tested with the CDC-NBA. IgG4 anti-FVIII antibodies were measured by fluorescence immunoassay (FLI). Results Diluted inhibitors showed linearity below 0.4 Nijmegen-Bethesda units (NBU). With four statistical methods, the limit of detection of the CDC-NBA was determined to be 0.2 NBU. IgG4 anti-FVIII antibodies, which correlate most strongly with functional inhibitors, were present at rates above the background rate of healthy controls in specimens with titers ≥ 0.2 NBU and showed an increase in frequency from 14.3% at 0.4 NBU to 67% at the established threshold for positivity of 0.5 NBU. Conclusions The CDC-NBA can detect inhibitors down to 0.2 NBU. The FLI, which is more sensitive, demonstrates anti-FVIII IgG4 in some patients with negative (< 0.5) NBU. The sharp increase

  6. Novel chemo-enzymatic oligomers of cinnamic acids as direct and indirect inhibitors of coagulation proteinases.

    PubMed

    Monien, Bernhard H; Henry, Brian L; Raghuraman, Arjun; Hindle, Michael; Desai, Umesh R

    2006-12-01

    Thrombin and factor Xa, two important procoagulant enzymes, have been prime targets for regulation of clotting through the direct and indirect mechanism of inhibition. Our efforts on exploiting the indirect mechanism led us to study a carboxylic acid-based scaffold, which displayed major acceleration in the inhibition of these enzymes [J. Med. Chem.2005, 48, 1269, 5360]. This work advances the study to chemo-enzymatically prepared oligomers of 4-hydroxycinnamic acids, DHPs, which display interesting anticoagulant properties. Oligomers, ranging in size from tetramers to pentadecamers, were prepared through peroxidase-catalyzed oxidative coupling of caffeic, ferulic, and sinapic acids, and sulfated using triethylamine-sulfur trioxide complex. Chromatographic, spectroscopic, and elemental studies suggest that the DHPs are heterogeneous, polydisperse preparations composed of inter-monomer linkages similar to those found in natural lignins. Measurement of activated thromboplastin and prothrombin time indicates that both the sulfated and unsulfated derivatives of the DHPs display anticoagulant activity, which is dramatically higher than that of the reference polyacrylic acids. More interestingly, this activity approaches that of low-molecular-weight heparin with the sulfated derivative showing approximately 2- to 3-fold greater potency than the unsulfated parent. Studies on the inhibition of factor Xa and thrombin indicate that the oligomers exert their anticoagulant effect through both direct and indirect inhibition mechanisms. This dual inhibition property of 4-hydroxycinnamic acid-based DHP oligomers is the first example in inhibitors of coagulation. This work puts forward a novel, non-heparin structure, which may be exploited for the design of potent, dual action inhibitors of coagulation through combinatorial virtual screening on a library of DHP oligomers.

  7. Tissue factor-driven thrombin generation and inflammation in atherosclerosis.

    PubMed

    ten Cate, Hugo

    2012-05-01

    The transmembrane receptor tissue factor is a prominent protein expressed at macrophages and smooth muscle cells within human atherosclerotic lesions. While many coagulation proteins are detectable in atherosclerosis, a locally active thrombin and fibrin generating molecular machinery may be instrumental in manipulating cellular functions involved in atherogenesis. These include inflammation, angiogenesis and cell proliferation. Indeed, many experimental studies in mice show a correlation between hypercoagulability and increased atherosclerosis. In mice, the amount of atherosclerosis and/or the plaque phenotype, appear to be modifiable by specific anticoagulant interventions. While attempts to vary tissue factor level in the vasculature does not directly reduce plaque burden, the overexpression of tissue factor pathway inhibitor attenuates thrombogenicity and neo intima formation in mice. Moreover, inhibition of factor Xa or thrombin with novel selective agents, including rivaroxaban and dabigatran, inhibits inflammation associated with atherosclerosis in apoE(-/-) mice. The potential to modify a complex chronic disease like atherosclerosis with novel selective anticoagulants merits further clinical study.

  8. Gene silencing using the recessive rice bacterial blight resistance gene xa13 as a new paradigm in plant breeding.

    PubMed

    Li, Changyan; Wei, Jing; Lin, Yongjun; Chen, Hao

    2012-05-01

    Resistant germplasm resources are valuable for developing resistant varieties in agricultural production. However, recessive resistance genes are usually overlooked in hybrid breeding. Compared with dominant traits, however, they may confer resistance to different pathogenic races or pest biotypes with different mechanisms of action. The recessive rice bacterial blight resistance gene xa13, also involved in pollen development, has been cloned and its resistance mechanism has been recently characterized. This report describes the conversion of bacterial blight resistance mediated by the recessive xa13 gene into a dominant trait to facilitate its use in a breeding program. This was achieved by knockdown of the corresponding dominant allele Xa13 in transgenic rice using recently developed artificial microRNA technology. Tissue-specific promoters were used to exclude most of the expression of artificial microRNA in the anther to ensure that Xa13 functioned normally during pollen development. A battery of highly bacterial blight resistant transgenic plants with normal seed setting rates were acquired, indicating that highly specific gene silencing had been achieved. Our success with xa13 provides a paradigm that can be adapted to other recessive resistance genes.

  9. The rice immune receptor XA21 recognizes a tyrosine-sulfated protein from a Gram-negative bacterium

    PubMed Central

    Pruitt, Rory N.; Schwessinger, Benjamin; Joe, Anna; Thomas, Nicholas; Liu, Furong; Albert, Markus; Robinson, Michelle R.; Chan, Leanne Jade G.; Luu, Dee Dee; Chen, Huamin; Bahar, Ofir; Daudi, Arsalan; De Vleesschauwer, David; Caddell, Daniel; Zhang, Weiguo; Zhao, Xiuxiang; Li, Xiang; Heazlewood, Joshua L.; Ruan, Deling; Majumder, Dipali; Chern, Mawsheng; Kalbacher, Hubert; Midha, Samriti; Patil, Prabhu B.; Sonti, Ramesh V.; Petzold, Christopher J.; Liu, Chang C.; Brodbelt, Jennifer S.; Felix, Georg; Ronald, Pamela C.

    2015-01-01

    Surveillance of the extracellular environment by immune receptors is of central importance to eukaryotic survival. The rice receptor kinase XA21, which confers robust resistance to most strains of the Gram-negative bacterium Xanthomonas oryzae pv. oryzae (Xoo), is representative of a large class of cell surface immune receptors in plants and animals. We report the identification of a previously undescribed Xoo protein, called RaxX, which is required for activation of XA21-mediated immunity. Xoo strains that lack RaxX, or carry mutations in the single RaxX tyrosine residue (Y41), are able to evade XA21-mediated immunity. Y41 of RaxX is sulfated by the prokaryotic tyrosine sulfotransferase RaxST. Sulfated, but not nonsulfated, RaxX triggers hallmarks of the plant immune response in an XA21-dependent manner. A sulfated, 21–amino acid synthetic RaxX peptide (RaxX21-sY) is sufficient for this activity. Xoo field isolates that overcome XA21-mediated immunity encode an alternate raxX allele, suggesting that coevolutionary interactions between host and pathogen contribute to RaxX diversification. RaxX is highly conserved in many plant pathogenic Xanthomonas species. The new insights gained from the discovery and characterization of the sulfated protein, RaxX, can be applied to the development of resistant crop varieties and therapeutic reagents that have the potential to block microbial infection of both plants and animals. PMID:26601222

  10. The Use of a Dexamethasone-inducible System to Synchronize Xa21 Expression to Study Rice Immunity

    PubMed Central

    Caddell, Daniel F.; Wei, Tong; Park, Chang-Jin; Ronald, Pamela C.

    2016-01-01

    Inducible gene expression systems offer researchers the opportunity to synchronize target gene expression at particular developmental stages and in particular tissues. The glucocorticoid receptor (GR), a vertebrate steroid receptor, has been well adopted for this purpose in plants. To generate steroid-inducible plants, a construct of GAL4-binding domain-VP16 activation domain-GR fusion (GVG) with the target gene under the control of upstream activation sequence (UAS) has been developed and extensively used in plant research. Immune receptors perceive conserved molecular patterns secreted by pathogens and initiate robust immune responses. The rice immune receptor, XA21, recognizes a molecular pattern highly conserved in all sequenced genomes of Xanthomonas, and confers robust resistance to X. oryzae pv. oryzae (Xoo). However, identifying genes downstream of XA21 has been hindered because of the restrained lesion and thus limited defense response region in the plants expressing Xa21. Inducible expression allows for a synchronized immune response across a large amount of rice tissue, well suited for studying XA21-mediated immunity by genome-wide approaches such as transcriptomics and proteomics. In this protocol, we describe the use of this GVG system to synchronize Xa21 expression. PMID:27525297

  11. Explicit polynomial formulas for solutions of the matrix equation AX-XA=C

    SciTech Connect

    Demenchuk, Alexandr K.; Makarov, Evgenii K.

    2009-08-15

    We provide some mathematical tool to analyze the possible theoretical structure of Hamiltonian reconstructed from von Neumann or Heisenberg's equation for a finite-dimensional quantum system. To this end, we give an explicit polynomial representation for the general solution of the matrix equation AX-XA=C together with some (also polynomial) solvability conditions when A and C are some complex square matrices of the same size and the matrix A is semisimple. When the matrix A is normal, we derive a polynomial existence condition for Hermitian solutions and a similar formula for all solutions of this type.

  12. Use of mTOR inhibitors in the treatment of breast cancer: an evaluation of factors that influence patient outcomes

    PubMed Central

    Jerusalem, Guy; Rorive, Andree; Collignon, Joelle

    2014-01-01

    Many systemic treatment options are available for advanced breast cancer, including endocrine therapy, chemotherapy, anti-human epidermal growth factor receptor 2 (HER2) therapy, and other targeted agents. Recently, everolimus, a mammalian target of rapamycin (mTOR) inhibitor, combined with exemestane, an aromatase inhibitor, has been approved in Europe and the USA for patients suffering from estrogen receptor-positive, HER2-negative advanced breast cancer previously treated by a nonsteroidal aromatase inhibitor, based on the results of BOLERO-2 (Breast cancer trials of OraL EveROlimus). This study showed a statistically significant and clinically meaningful improvement in median progression-free survival. Results concerning the impact on overall survival are expected in the near future. This clinically oriented review focuses on the use of mTOR inhibitors in breast cancer. Results reported with first-generation mTOR inhibitors (ridaforolimus, temsirolimus, everolimus) are discussed. The current and potential role of mTOR inhibitors is reported according to breast cancer subtype (estrogen receptor-positive HER2-negative, triple-negative, and HER2-positive ER-positive/negative disease). Everolimus is currently being evaluated in the adjuvant setting in high-risk estrogen receptor-positive, HER2-negative early breast cancer. Continuing mTOR inhibition or alternatively administering other drugs targeting the phosphatidylinositol-3-kinase/protein kinase B-mTOR pathway after progression on treatments including an mTOR inhibitor is under evaluation. Potential biomarkers to select patients showing a more pronounced benefit are reviewed, but we are not currently using these biomarkers in routine practice. Subgroup analysis of BOLERO 2 has shown that the benefit is consistent in all subgroups and that it is impossible to select patients not benefiting from addition of everolimus to exemestane. Side effects and impact on quality of life are other important issues discussed

  13. Acquired factor V inhibitor in a patient receiving venous-venous extracorporeal membrane oxygenation for Legionella pneumonia.

    PubMed

    Leung, Anne K H; Ng, George W Y; Sin, K C; Au, S Y; Lai, K Y; Lee, K L; Law, K I

    2015-04-01

    We report a rare complication of factor V deficiency in a patient having Legionella pneumonia. This patient also had other complications like severe acute respiratory distress syndrome,